1645_ftp

7/25/2019 1645_ftp

1/16

See discussions, stats, and author profiles for this publication at: https://www.researchgate.net/publication/23960687

Ubiquitin ligase Rsp5p is involved in the

gene expression changes during nutrient

limitation in Saccharomyces cerevisiae

ARTICLE inYEAST JANUARY 2009

Impact Factor: 1.63 DOI: 10.1002/yea.1645 Source: PubMed

CITATIONS

8

READS

21

3 AUTHORS:

Fernando Cardona

Spanish National Research Council

15PUBLICATIONS 74CITATIONS

SEE PROFILE

Agustn Aranda

Spanish National Research Council


SEE PROFILE

Marcell del Olmo

University of Valencia


SEE PROFILE

All in-text references underlined in blueare linked to publications on ResearchGate,

letting you access and read them immediately.

Available from: Fernando Cardona

Retrieved on: 19 January 2016
https://www.researchgate.net/profile/Marcelli_Olmo?enrichId=rgreq-3066aad4-419b-48b3-a8da-2929dbaaf062&enrichSource=Y292ZXJQYWdlOzIzOTYwNjg3O0FTOjEwMzc5MjQ5MDA1NzczNUAxNDAxNzU3NDY2MzI4&el=1_x_4https://www.researchgate.net/institution/University_of_Valencia?enrichId=rgreq-3066aad4-419b-48b3-a8da-2929dbaaf062&enrichSource=Y292ZXJQYWdlOzIzOTYwNjg3O0FTOjEwMzc5MjQ5MDA1NzczNUAxNDAxNzU3NDY2MzI4&el=1_x_6https://www.researchgate.net/profile/Fernando_Cardona2?enrichId=rgreq-3066aad4-419b-48b3-a8da-2929dbaaf062&enrichSource=Y292ZXJQYWdlOzIzOTYwNjg3O0FTOjEwMzc5MjQ5MDA1NzczNUAxNDAxNzU3NDY2MzI4&el=1_x_4https://www.researchgate.net/publication/23960687_Ubiquitin_ligase_Rsp5p_is_involved_in_the_gene_expression_changes_during_nutrient_limitation_in_Saccharomyces_cerevisiae?enrichId=rgreq-3066aad4-419b-48b3-a8da-2929dbaaf062&enrichSource=Y292ZXJQYWdlOzIzOTYwNjg3O0FTOjEwMzc5MjQ5MDA1NzczNUAxNDAxNzU3NDY2MzI4&el=1_x_3https://www.researchgate.net/publication/23960687_Ubiquitin_ligase_Rsp5p_is_involved_in_the_gene_expression_changes_during_nutrient_limitation_in_Saccharomyces_cerevisiae?enrichId=rgreq-3066aad4-419b-48b3-a8da-2929dbaaf062&enrichSource=Y292ZXJQYWdlOzIzOTYwNjg3O0FTOjEwMzc5MjQ5MDA1NzczNUAxNDAxNzU3NDY2MzI4&el=1_x_3https://www.researchgate.net/publication/23960687_Ubiquitin_ligase_Rsp5p_is_involved_in_the_gene_expression_changes_during_nutrient_limitation_in_Saccharomyces_cerevisiae?enrichId=rgreq-3066aad4-419b-48b3-a8da-2929dbaaf062&enrichSource=Y292ZXJQYWdlOzIzOTYwNjg3O0FTOjEwMzc5MjQ5MDA1NzczNUAxNDAxNzU3NDY2MzI4&el=1_x_3https://www.researchgate.net/publication/23960687_Ubiquitin_ligase_Rsp5p_is_involved_in_the_gene_expression_changes_during_nutrient_limitation_in_Saccharomyces_cerevisiae?enrichId=rgreq-3066aad4-419b-48b3-a8da-2929dbaaf062&enrichSource=Y292ZXJQYWdlOzIzOTYwNjg3O0FTOjEwMzc5MjQ5MDA1NzczNUAxNDAxNzU3NDY2MzI4&el=1_x_3https://www.researchgate.net/publication/23960687_Ubiquitin_ligase_Rsp5p_is_involved_in_the_gene_expression_changes_during_nutrient_limitation_in_Saccharomyces_cerevisiae?enrichId=rgreq-3066aad4-419b-48b3-a8da-2929dbaaf062&enrichSource=Y292ZXJQYWdlOzIzOTYwNjg3O0FTOjEwMzc5MjQ5MDA1NzczNUAxNDAxNzU3NDY2MzI4&el=1_x_3https://www.researchgate.net/publication/23960687_Ubiquitin_ligase_Rsp5p_is_involved_in_the_gene_expression_changes_during_nutrient_limitation_in_Saccharomyces_cerevisiae?enrichId=rgreq-3066aad4-419b-48b3-a8da-2929dbaaf062&enrichSource=Y292ZXJQYWdlOzIzOTYwNjg3O0FTOjEwMzc5MjQ5MDA1NzczNUAxNDAxNzU3NDY2MzI4&el=1_x_3https://www.researchgate.net/publication/23960687_Ubiquitin_ligase_Rsp5p_is_involved_in_the_gene_expression_changes_during_nutrient_limitation_in_Saccharomyces_cerevisiae?enrichId=rgreq-3066aad4-419b-48b3-a8da-2929dbaaf062&enrichSource=Y292ZXJQYWdlOzIzOTYwNjg3O0FTOjEwMzc5MjQ5MDA1NzczNUAxNDAxNzU3NDY2MzI4&el=1_x_3https://www.researchgate.net/publication/23960687_Ubiquitin_ligase_Rsp5p_is_involved_in_the_gene_expression_changes_during_nutrient_limitation_in_Saccharomyces_cerevisiae?enrichId=rgreq-3066aad4-419b-48b3-a8da-2929dbaaf062&enrichSource=Y292ZXJQYWdlOzIzOTYwNjg3O0FTOjEwMzc5MjQ5MDA1NzczNUAxNDAxNzU3NDY2MzI4&el=1_x_3https://www.researchgate.net/publication/23960687_Ubiquitin_ligase_Rsp5p_is_involved_in_the_gene_expression_changes_during_nutrient_limitation_in_Saccharomyces_cerevisiae?enrichId=rgreq-3066aad4-419b-48b3-a8da-2929dbaaf062&enrichSource=Y292ZXJQYWdlOzIzOTYwNjg3O0FTOjEwMzc5MjQ5MDA1NzczNUAxNDAxNzU3NDY2MzI4&el=1_x_3https://www.researchgate.net/publication/23960687_Ubiquitin_ligase_Rsp5p_is_involved_in_the_gene_expression_changes_during_nutrient_limitation_in_Saccharomyces_cerevisiae?enrichId=rgreq-3066aad4-419b-48b3-a8da-2929dbaaf062&enrichSource=Y292ZXJQYWdlOzIzOTYwNjg3O0FTOjEwMzc5MjQ5MDA1NzczNUAxNDAxNzU3NDY2MzI4&el=1_x_3https://www.researchgate.net/publication/23960687_Ubiquitin_ligase_Rsp5p_is_involved_in_the_gene_expression_changes_during_nutrient_limitation_in_Saccharomyces_cerevisiae?enrichId=rgreq-3066aad4-419b-48b3-a8da-2929dbaaf062&enrichSource=Y292ZXJQYWdlOzIzOTYwNjg3O0FTOjEwMzc5MjQ5MDA1NzczNUAxNDAxNzU3NDY2MzI4&el=1_x_3https://www.researchgate.net/?enrichId=rgreq-3066aad4-419b-48b3-a8da-2929dbaaf062&enrichSource=Y292ZXJQYWdlOzIzOTYwNjg3O0FTOjEwMzc5MjQ5MDA1NzczNUAxNDAxNzU3NDY2MzI4&el=1_x_1https://www.researchgate.net/profile/Marcelli_Olmo?enrichId=rgreq-3066aad4-419b-48b3-a8da-2929dbaaf062&enrichSource=Y292ZXJQYWdlOzIzOTYwNjg3O0FTOjEwMzc5MjQ5MDA1NzczNUAxNDAxNzU3NDY2MzI4&el=1_x_7https://www.researchgate.net/institution/University_of_Valencia?enrichId=rgreq-3066aad4-419b-48b3-a8da-2929dbaaf062&enrichSource=Y292ZXJQYWdlOzIzOTYwNjg3O0FTOjEwMzc5MjQ5MDA1NzczNUAxNDAxNzU3NDY2MzI4&el=1_x_6https://www.researchgate.net/profile/Marcelli_Olmo?enrichId=rgreq-3066aad4-419b-48b3-a8da-2929dbaaf062&enrichSource=Y292ZXJQYWdlOzIzOTYwNjg3O0FTOjEwMzc5MjQ5MDA1NzczNUAxNDAxNzU3NDY2MzI4&el=1_x_5https://www.researchgate.net/profile/Marcelli_Olmo?enrichId=rgreq-3066aad4-419b-48b3-a8da-2929dbaaf062&enrichSource=Y292ZXJQYWdlOzIzOTYwNjg3O0FTOjEwMzc5MjQ5MDA1NzczNUAxNDAxNzU3NDY2MzI4&el=1_x_4https://www.researchgate.net/profile/Agustin_Aranda?enrichId=rgreq-3066aad4-419b-48b3-a8da-2929dbaaf062&enrichSource=Y292ZXJQYWdlOzIzOTYwNjg3O0FTOjEwMzc5MjQ5MDA1NzczNUAxNDAxNzU3NDY2MzI4&el=1_x_7https://www.researchgate.net/institution/Spanish_National_Research_Council?enrichId=rgreq-3066aad4-419b-48b3-a8da-2929dbaaf062&enrichSource=Y292ZXJQYWdlOzIzOTYwNjg3O0FTOjEwMzc5MjQ5MDA1NzczNUAxNDAxNzU3NDY2MzI4&el=1_x_6https://www.researchgate.net/profile/Agustin_Aranda?enrichId=rgreq-3066aad4-419b-48b3-a8da-2929dbaaf062&enrichSource=Y292ZXJQYWdlOzIzOTYwNjg3O0FTOjEwMzc5MjQ5MDA1NzczNUAxNDAxNzU3NDY2MzI4&el=1_x_5https://www.researchgate.net/profile/Agustin_Aranda?enrichId=rgreq-3066aad4-419b-48b3-a8da-2929dbaaf062&enrichSource=Y292ZXJQYWdlOzIzOTYwNjg3O0FTOjEwMzc5MjQ5MDA1NzczNUAxNDAxNzU3NDY2MzI4&el=1_x_4https://www.researchgate.net/profile/Fernando_Cardona2?enrichId=rgreq-3066aad4-419b-48b3-a8da-2929dbaaf062&enrichSource=Y292ZXJQYWdlOzIzOTYwNjg3O0FTOjEwMzc5MjQ5MDA1NzczNUAxNDAxNzU3NDY2MzI4&el=1_x_7https://www.researchgate.net/institution/Spanish_National_Research_Council?enrichId=rgreq-3066aad4-419b-48b3-a8da-2929dbaaf062&enrichSource=Y292ZXJQYWdlOzIzOTYwNjg3O0FTOjEwMzc5MjQ5MDA1NzczNUAxNDAxNzU3NDY2MzI4&el=1_x_6https://www.researchgate.net/profile/Fernando_Cardona2?enrichId=rgreq-3066aad4-419b-48b3-a8da-2929dbaaf062&enrichSource=Y292ZXJQYWdlOzIzOTYwNjg3O0FTOjEwMzc5MjQ5MDA1NzczNUAxNDAxNzU3NDY2MzI4&el=1_x_5https://www.researchgate.net/profile/Fernando_Cardona2?enrichId=rgreq-3066aad4-419b-48b3-a8da-2929dbaaf062&enrichSource=Y292ZXJQYWdlOzIzOTYwNjg3O0FTOjEwMzc5MjQ5MDA1NzczNUAxNDAxNzU3NDY2MzI4&el=1_x_4https://www.researchgate.net/?enrichId=rgreq-3066aad4-419b-48b3-a8da-2929dbaaf062&enrichSource=Y292ZXJQYWdlOzIzOTYwNjg3O0FTOjEwMzc5MjQ5MDA1NzczNUAxNDAxNzU3NDY2MzI4&el=1_x_1https://www.researchgate.net/publication/23960687_Ubiquitin_ligase_Rsp5p_is_involved_in_the_gene_expression_changes_during_nutrient_limitation_in_Saccharomyces_cerevisiae?enrichId=rgreq-3066aad4-419b-48b3-a8da-2929dbaaf062&enrichSource=Y292ZXJQYWdlOzIzOTYwNjg3O0FTOjEwMzc5MjQ5MDA1NzczNUAxNDAxNzU3NDY2MzI4&el=1_x_3https://www.researchgate.net/publication/23960687_Ubiquitin_ligase_Rsp5p_is_involved_in_the_gene_expression_changes_during_nutrient_limitation_in_Saccharomyces_cerevisiae?enrichId=rgreq-3066aad4-419b-48b3-a8da-2929dbaaf062&enrichSource=Y292ZXJQYWdlOzIzOTYwNjg3O0FTOjEwMzc5MjQ5MDA1NzczNUAxNDAxNzU3NDY2MzI4&el=1_x_2

7/25/2019 1645_ftp

2/16

Yeast

Yeast2009;26: 115.

Published online in Wiley InterScience

(www.interscience.wiley.com)DOI:10.1002/yea.1645

Research Article

Ubiquitin ligase Rsp5p is involved in the gene

expression changes during nutrient limitationinSaccharomyces cerevisiae

F. Cardona1,2#, A. Aranda1,2*and M. del Olmo1

1Department of Biochemistry and Molecular Biology, University of Valencia, Spain2Department of Biotechnology, IATA (CSIC), Valencia, Spain

*Correspondence to:A. Aranda, Departament deBioqumica i Biologia Molecular,Universitat de Valencia, Apartado73, Burjassot, Valencia

46100, Spain.E-mail: [email protected]

#Present address: Unitat deGenetica Molecular, Institut deBiomedicina de Valencia (CSIC),Valencia, Spain.

Received: 5 June 2008

Accepted: 21 October 2008

Abstract

Rsp5p is an essential ubiquitin ligase involved in many different cellular events,

including amino acid transporters degradation, transcription initiation and mRNA

export. It plays important role in both stress resistance and adaptation to the change

of nutrients. We have found that ubiquitination machinery is necessary for the correctinduction of the stress response SPI1 gene at the entry of the stationary phase. SPI1

is a gene whose expression is regulated by the nutritional status of the cell and whose

deletion causes hypersensitivity to various stresses, such as heat shock, alkaline stress

and oxidative stress. Its regulation is mastered by Rsp5p, as mutations in this gene

lead to a lower SPI1 expression. In this process, Rsp5p is helped by several proteins,

such as Rsp5p-interacting proteins Bul1p/2p, the ubiquitin conjugating protein Ubc1p

and ubiquitin proteases Ubp4p and Ubp16p. Moreover, a mutation in the RSP5 gene

has a global effect at the gene expression level when cells enter the stationary phase.

Rsp5p particularly controls the levels of the ribosomal proteins mRNAs at this stage.

Rsp5p is also necessary for a correct induction of p-bodies under stress conditions,

indicating that this protein plays an important role in the post-transcriptional fate of

mRNA under nutrient starvation. Copyright 2009 John Wiley & Sons, Ltd.

Keywords: RSP5; SPI1 ; stress response; stationary phase; p-bodies

Introduction

Nutrient depletion in Saccharomyces cerevisiaeinduces a plethora of physiological, biochemicaland morphological changes. A yeast culture grownon glucose-based medium changes from a ferment-ing metabolism to a respiratory one when glucosebecomes limited. This phenomenon is called thediauxic shift. When the carbon source is exhausted,the culture enters the so-called stationary phase(Gray et al., 2004; Herman, 2002). This stateallows cell survival over long periods of time with-out added nutrients. Cells in stationary phase cul-tures accumulate glycogen and trehalose, develop athickened cell wall and become resistant to stressessuch as increased temperature and oxidative stress.

Entry into the stationary phase has been describedunder laboratory conditions (Grayet al., 2004; Her-man, 2002). The target of rapamycin kinase (TOR)and protein kinase A (PKA) transduction signalpathways coordinately respond to nutrient avail-ability, thus favouring growth and cellular divisionand repressing the stress response (Jorgensenet al.,

2004; Martin and Hall, 2005). One of the gene fam-ilies involved in cell growth that is regulated bythese pathways and depends on nutrient availabil-ity is the ribosomal proteins (RP) (Jorgensen et al.,2004; Powers, 2004). On the other hand, thesepathways under nutrient depletion block the cellcycle and allow the activation of stress responsemechanisms (Hohman and Mager, 2003, and ref-erences therein). Several reports have described

Copyright 2009 John Wiley & Sons, Ltd.
https://www.researchgate.net/publication/11011982_Stationary_phase_in_yeast?el=1_x_8&enrichId=rgreq-3066aad4-419b-48b3-a8da-2929dbaaf062&enrichSource=Y292ZXJQYWdlOzIzOTYwNjg3O0FTOjEwMzc5MjQ5MDA1NzczNUAxNDAxNzU3NDY2MzI4https://www.researchgate.net/publication/11011982_Stationary_phase_in_yeast?el=1_x_8&enrichId=rgreq-3066aad4-419b-48b3-a8da-2929dbaaf062&enrichSource=Y292ZXJQYWdlOzIzOTYwNjg3O0FTOjEwMzc5MjQ5MDA1NzczNUAxNDAxNzU3NDY2MzI4https://www.researchgate.net/publication/11011982_Stationary_phase_in_yeast?el=1_x_8&enrichId=rgreq-3066aad4-419b-48b3-a8da-2929dbaaf062&enrichSource=Y292ZXJQYWdlOzIzOTYwNjg3O0FTOjEwMzc5MjQ5MDA1NzczNUAxNDAxNzU3NDY2MzI4https://www.researchgate.net/publication/11011982_Stationary_phase_in_yeast?el=1_x_8&enrichId=rgreq-3066aad4-419b-48b3-a8da-2929dbaaf062&enrichSource=Y292ZXJQYWdlOzIzOTYwNjg3O0FTOjEwMzc5MjQ5MDA1NzczNUAxNDAxNzU3NDY2MzI4

7/25/2019 1645_ftp

3/16

2 F. Cardona, A. Aranda and M. del Olmo

extensive transcriptomic changes under stationaryphase conditions (DeRisi et al., 1997; Gasch et al.,2000). Recently the existence of a large number ofextraction-resistant mRNAs in the stationary phasehas been described (Aragon et al., 2006). Thesetranscripts are protein-bound, as they are extracted

by protease action and are rapidly released after astressful condition such as oxidative shock. Understress conditions, such as nutrient deprivation, thisphenomenon has been linked to the induction ofmRNA relocalization to sites of degradation orstorage of RNA called processing bodies (p-bodies)(Parker and Sheth, 2007). RNAs accumulated inthese protein-bound structures can return to trans-lation when the stress disappears (Brengues et al.,2005). Therefore, they are a post-transcriptionalway to regulate the gene expression.

Ubiquitination is a complex protein modification

process that involves a cascade of E1 (ubiquitin-activating), E2 (ubiquitin-conjugating) and E3(ubiquitin ligases) enzymes which regulate ubiq-uitination specificity (Pickart, 2004). Polyubiquitinchains are built through the K29 and K48 residuesof ubiquitin target proteins for proteasomal degra-dation by the 26S proteasome(Hochstrasser, 1996),whereas polyubiquitin K63 chains are associatedwith stress resistance, DNA repair, signal transduc-tion, and degradation via the vacuole (Horak, 2003;Sigismund et al., 2004). Mono-ubiquitination atK63 controls many processes, including mem-

brane trafficking, meiosis and chromatin modelling(Hicke, 2001). Ubiquitinated proteins undergo de-ubiquitination by proteases called de-ubiquitinatingenzymes (DUBs) prior to degradation (Pickart,2004). The yeast HECT-domain ubiquitin-ligaseRsp5p, which adds mono- and polyubiquitin chainslinked through K63, has been involved in awide variety of physiological processes, such asminichromosome and actin cytoskeleton mainte-nance, mitochondrial inheritance, drug resistance,regulation of intracellular pH, biosynthesis of fattyacids, cell wall organization and protein sorting(Horak, 2003; Kaminska et al., 2005; Kus et al.,2005). Recent studies have linked Rsp5p to allthe levels of gene expression. The activity of tran-scription factor Spt23p is specifically activated byRsp5p-mediated proteolysis (Hoppe et al., 2000).At the general transcription machinery level, Rsp5pinteracts with RNA polymerase II CTD and stim-ulates its phosphorylation (Max et al., 2007) andubiquitination, as well as the destruction of RNA

pol IIin response to DNA damage (Somesh et al.,2005). The export of all three kinds of RNArequires Rsp5p (Neumann et al., 2003) and alsoHpr1p, a member of the THO/TREX (transcrip-tion/export) complex, which is regulated by Rsp5-mediated ubiquitination (Gwizdek et al., 2005).

Finally, recent data indicate an important role of theRsp5p-mediated ubiquitination in translation accu-racy(Kwapisz et al., 2006).

There are evidences that links Rsp5p to thestress response in S. cerevisiae. Rsp5p binds totwo homologous proteins, Bul1p and Bul2p, andthe double disruptant bul1bul2 is sensitive to var-

ious stresses, including a growth defect on anon-fermentable carbon source (Yashiroda et al.,1998). The Rsp5pBul1pBul2p complex is nec-

essary for heat shock element (HSE)-mediated tran-scription (Kaida et al. 2003). This ubiquitin lig-ase has been described to regulate the expression

of stress proteins via post-translational modifica-tion of the stress response transcription factorsHsf1p and Msn4p (Haitani et al., 2006). Recentlyit has been shown that Rsp5p is required for thenuclear export of HSF1 and MSN2/4 mRNAsunder stress conditions(Haitani and Takagi, 2008).Besides, cells overexpressing Rsp5p or ubiquitin-conjugation enzymes (E2, Ubc1Ubc13) displayenhanced tolerance to several forms of stress (heatshock, osmotic stress, oxidative stress and ethanol)

(Hiraishiet al., 2006).We are interested in the transcriptional response

at postdiauxic and stationary phases and we haveused SPI1, one of the yeast genes induced underthese conditions during industrial (Puig and Perez-Ortn, 2000) and laboratory conditions (Gaschet al., 2000), as a model. Its transcription also risesunder several stress conditions (Gaschet al., 2000).It encodes a protein which is important for thestructure and biogenesis of the cell wall (Horieand Isono, 2001). In this study we analysed therelevance of SPI1 under several stress conditions

and we particularly focused on its response duringentrance into the stationary phase. We showedthat the expression of this gene is affected by

not only the ubiquitin-ligase Rsp5p but also someinteracting proteins, and we provide evidence ofthis proteins involvement in the tolerance to this

and to other forms of stress. Furthermore, we havefound that Rsp5p represses the expression levelsof ribosomal proteins (RPs) at the start of the

Copyright 2009 John Wiley & Sons, Ltd. Yeast2009; 26: 115.

DOI: 10.1002/yea
https://www.researchgate.net/publication/6456944_P_Bodies_and_the_Control_of_mRNA_Translation_and_Degradation_Mol_Cell?el=1_x_8&enrichId=rgreq-3066aad4-419b-48b3-a8da-2929dbaaf062&enrichSource=Y292ZXJQYWdlOzIzOTYwNjg3O0FTOjEwMzc5MjQ5MDA1NzczNUAxNDAxNzU3NDY2MzI4https://www.researchgate.net/publication/14226568_Hochstrasser_MUbiquitin-dependent_protein_degradation_Ann_Rev_Genet_30_405-439?el=1_x_8&enrichId=rgreq-3066aad4-419b-48b3-a8da-2929dbaaf062&enrichSource=Y292ZXJQYWdlOzIzOTYwNjg3O0FTOjEwMzc5MjQ5MDA1NzczNUAxNDAxNzU3NDY2MzI4https://www.researchgate.net/publication/12068182_Protein_regulation_by_monoubiquitin?el=1_x_8&enrichId=rgreq-3066aad4-419b-48b3-a8da-2929dbaaf062&enrichSource=Y292ZXJQYWdlOzIzOTYwNjg3O0FTOjEwMzc5MjQ5MDA1NzczNUAxNDAxNzU3NDY2MzI4https://www.researchgate.net/publication/6430061_Hyperphosphorylation_of_the_C-terminal_Repeat_Domain_of_RNA_Polymerase_II_Facilitates_Dissociation_of_Its_Complex_with_Mediator?el=1_x_8&enrichId=rgreq-3066aad4-419b-48b3-a8da-2929dbaaf062&enrichSource=Y292ZXJQYWdlOzIzOTYwNjg3O0FTOjEwMzc5MjQ5MDA1NzczNUAxNDAxNzU3NDY2MzI4https://www.researchgate.net/publication/6430061_Hyperphosphorylation_of_the_C-terminal_Repeat_Domain_of_RNA_Polymerase_II_Facilitates_Dissociation_of_Its_Complex_with_Mediator?el=1_x_8&enrichId=rgreq-3066aad4-419b-48b3-a8da-2929dbaaf062&enrichSource=Y292ZXJQYWdlOzIzOTYwNjg3O0FTOjEwMzc5MjQ5MDA1NzczNUAxNDAxNzU3NDY2MzI4https://www.researchgate.net/publication/6430061_Hyperphosphorylation_of_the_C-terminal_Repeat_Domain_of_RNA_Polymerase_II_Facilitates_Dissociation_of_Its_Complex_with_Mediator?el=1_x_8&enrichId=rgreq-3066aad4-419b-48b3-a8da-2929dbaaf062&enrichSource=Y292ZXJQYWdlOzIzOTYwNjg3O0FTOjEwMzc5MjQ5MDA1NzczNUAxNDAxNzU3NDY2MzI4https://www.researchgate.net/publication/8018823_The_mRNA_Nuclear_Export_Factor_Hpr1_Is_Regulated_by_Rsp5-mediated_Ubiquitylation?el=1_x_8&enrichId=rgreq-3066aad4-419b-48b3-a8da-2929dbaaf062&enrichSource=Y292ZXJQYWdlOzIzOTYwNjg3O0FTOjEwMzc5MjQ5MDA1NzczNUAxNDAxNzU3NDY2MzI4https://www.researchgate.net/publication/8018823_The_mRNA_Nuclear_Export_Factor_Hpr1_Is_Regulated_by_Rsp5-mediated_Ubiquitylation?el=1_x_8&enrichId=rgreq-3066aad4-419b-48b3-a8da-2929dbaaf062&enrichSource=Y292ZXJQYWdlOzIzOTYwNjg3O0FTOjEwMzc5MjQ5MDA1NzczNUAxNDAxNzU3NDY2MzI4https://www.researchgate.net/publication/8018823_The_mRNA_Nuclear_Export_Factor_Hpr1_Is_Regulated_by_Rsp5-mediated_Ubiquitylation?el=1_x_8&enrichId=rgreq-3066aad4-419b-48b3-a8da-2929dbaaf062&enrichSource=Y292ZXJQYWdlOzIzOTYwNjg3O0FTOjEwMzc5MjQ5MDA1NzczNUAxNDAxNzU3NDY2MzI4https://www.researchgate.net/publication/7587092_Rsp5_ubiquitin_ligase_modulates_translation_accuracy_in_yeast_Saccharomyces_cerevisiae?el=1_x_8&enrichId=rgreq-3066aad4-419b-48b3-a8da-2929dbaaf062&enrichSource=Y292ZXJQYWdlOzIzOTYwNjg3O0FTOjEwMzc5MjQ5MDA1NzczNUAxNDAxNzU3NDY2MzI4https://www.researchgate.net/publication/7587092_Rsp5_ubiquitin_ligase_modulates_translation_accuracy_in_yeast_Saccharomyces_cerevisiae?el=1_x_8&enrichId=rgreq-3066aad4-419b-48b3-a8da-2929dbaaf062&enrichSource=Y292ZXJQYWdlOzIzOTYwNjg3O0FTOjEwMzc5MjQ5MDA1NzczNUAxNDAxNzU3NDY2MzI4https://www.researchgate.net/publication/7587092_Rsp5_ubiquitin_ligase_modulates_translation_accuracy_in_yeast_Saccharomyces_cerevisiae?el=1_x_8&enrichId=rgreq-3066aad4-419b-48b3-a8da-2929dbaaf062&enrichSource=Y292ZXJQYWdlOzIzOTYwNjg3O0FTOjEwMzc5MjQ5MDA1NzczNUAxNDAxNzU3NDY2MzI4https://www.researchgate.net/publication/7067340_Rsp5_regulates_expression_of_stress_proteins_via_post-translational_modification_of_Hsf1_and_Msn4_in_Saccharomyces_cerevisiae?el=1_x_8&enrichId=rgreq-3066aad4-419b-48b3-a8da-2929dbaaf062&enrichSource=Y292ZXJQYWdlOzIzOTYwNjg3O0FTOjEwMzc5MjQ5MDA1NzczNUAxNDAxNzU3NDY2MzI4https://www.researchgate.net/publication/7067340_Rsp5_regulates_expression_of_stress_proteins_via_post-translational_modification_of_Hsf1_and_Msn4_in_Saccharomyces_cerevisiae?el=1_x_8&enrichId=rgreq-3066aad4-419b-48b3-a8da-2929dbaaf062&enrichSource=Y292ZXJQYWdlOzIzOTYwNjg3O0FTOjEwMzc5MjQ5MDA1NzczNUAxNDAxNzU3NDY2MzI4https://www.researchgate.net/publication/7067340_Rsp5_regulates_expression_of_stress_proteins_via_post-translational_modification_of_Hsf1_and_Msn4_in_Saccharomyces_cerevisiae?el=1_x_8&enrichId=rgreq-3066aad4-419b-48b3-a8da-2929dbaaf062&enrichSource=Y292ZXJQYWdlOzIzOTYwNjg3O0FTOjEwMzc5MjQ5MDA1NzczNUAxNDAxNzU3NDY2MzI4https://www.researchgate.net/publication/5617887_Rsp5_is_required_for_the_nuclear_export_of_mRNA_of_HSF1_and_MSN24_under_stress_conditions_in_Saccharomyces_cerevisiae?el=1_x_8&enrichId=rgreq-3066aad4-419b-48b3-a8da-2929dbaaf062&enrichSource=Y292ZXJQYWdlOzIzOTYwNjg3O0FTOjEwMzc5MjQ5MDA1NzczNUAxNDAxNzU3NDY2MzI4https://www.researchgate.net/publication/6706231_Enhancement_of_Stress_Tolerance_in_Saccharomyces_cerevisiae_by_Overexpression_of_Ubiquitin_Ligase_Rsp5_and_Ubiquitin-Conjugating_Enzymes?el=1_x_8&enrichId=rgreq-3066aad4-419b-48b3-a8da-2929dbaaf062&enrichSource=Y292ZXJQYWdlOzIzOTYwNjg3O0FTOjEwMzc5MjQ5MDA1NzczNUAxNDAxNzU3NDY2MzI4https://www.researchgate.net/publication/6706231_Enhancement_of_Stress_Tolerance_in_Saccharomyces_cerevisiae_by_Overexpression_of_Ubiquitin_Ligase_Rsp5_and_Ubiquitin-Conjugating_Enzymes?el=1_x_8&enrichId=rgreq-3066aad4-419b-48b3-a8da-2929dbaaf062&enrichSource=Y292ZXJQYWdlOzIzOTYwNjg3O0FTOjEwMzc5MjQ5MDA1NzczNUAxNDAxNzU3NDY2MzI4https://www.researchgate.net/publication/6706231_Enhancement_of_Stress_Tolerance_in_Saccharomyces_cerevisiae_by_Overexpression_of_Ubiquitin_Ligase_Rsp5_and_Ubiquitin-Conjugating_Enzymes?el=1_x_8&enrichId=rgreq-3066aad4-419b-48b3-a8da-2929dbaaf062&enrichSource=Y292ZXJQYWdlOzIzOTYwNjg3O0FTOjEwMzc5MjQ5MDA1NzczNUAxNDAxNzU3NDY2MzI4https://www.researchgate.net/publication/12068182_Protein_regulation_by_monoubiquitin?el=1_x_8&enrichId=rgreq-3066aad4-419b-48b3-a8da-2929dbaaf062&enrichSource=Y292ZXJQYWdlOzIzOTYwNjg3O0FTOjEwMzc5MjQ5MDA1NzczNUAxNDAxNzU3NDY2MzI4https://www.researchgate.net/publication/7587092_Rsp5_ubiquitin_ligase_modulates_translation_accuracy_in_yeast_Saccharomyces_cerevisiae?el=1_x_8&enrichId=rgreq-3066aad4-419b-48b3-a8da-2929dbaaf062&enrichSource=Y292ZXJQYWdlOzIzOTYwNjg3O0FTOjEwMzc5MjQ5MDA1NzczNUAxNDAxNzU3NDY2MzI4https://www.researchgate.net/publication/5617887_Rsp5_is_required_for_the_nuclear_export_of_mRNA_of_HSF1_and_MSN24_under_stress_conditions_in_Saccharomyces_cerevisiae?el=1_x_8&enrichId=rgreq-3066aad4-419b-48b3-a8da-2929dbaaf062&enrichSource=Y292ZXJQYWdlOzIzOTYwNjg3O0FTOjEwMzc5MjQ5MDA1NzczNUAxNDAxNzU3NDY2MzI4https://www.researchgate.net/publication/6430061_Hyperphosphorylation_of_the_C-terminal_Repeat_Domain_of_RNA_Polymerase_II_Facilitates_Dissociation_of_Its_Complex_with_Mediator?el=1_x_8&enrichId=rgreq-3066aad4-419b-48b3-a8da-2929dbaaf062&enrichSource=Y292ZXJQYWdlOzIzOTYwNjg3O0FTOjEwMzc5MjQ5MDA1NzczNUAxNDAxNzU3NDY2MzI4https://www.researchgate.net/publication/7067340_Rsp5_regulates_expression_of_stress_proteins_via_post-translational_modification_of_Hsf1_and_Msn4_in_Saccharomyces_cerevisiae?el=1_x_8&enrichId=rgreq-3066aad4-419b-48b3-a8da-2929dbaaf062&enrichSource=Y292ZXJQYWdlOzIzOTYwNjg3O0FTOjEwMzc5MjQ5MDA1NzczNUAxNDAxNzU3NDY2MzI4https://www.researchgate.net/publication/14226568_Hochstrasser_MUbiquitin-dependent_protein_degradation_Ann_Rev_Genet_30_405-439?el=1_x_8&enrichId=rgreq-3066aad4-419b-48b3-a8da-2929dbaaf062&enrichSource=Y292ZXJQYWdlOzIzOTYwNjg3O0FTOjEwMzc5MjQ5MDA1NzczNUAxNDAxNzU3NDY2MzI4https://www.researchgate.net/publication/6706231_Enhancement_of_Stress_Tolerance_in_Saccharomyces_cerevisiae_by_Overexpression_of_Ubiquitin_Ligase_Rsp5_and_Ubiquitin-Conjugating_Enzymes?el=1_x_8&enrichId=rgreq-3066aad4-419b-48b3-a8da-2929dbaaf062&enrichSource=Y292ZXJQYWdlOzIzOTYwNjg3O0FTOjEwMzc5MjQ5MDA1NzczNUAxNDAxNzU3NDY2MzI4https://www.researchgate.net/publication/8018823_The_mRNA_Nuclear_Export_Factor_Hpr1_Is_Regulated_by_Rsp5-mediated_Ubiquitylation?el=1_x_8&enrichId=rgreq-3066aad4-419b-48b3-a8da-2929dbaaf062&enrichSource=Y292ZXJQYWdlOzIzOTYwNjg3O0FTOjEwMzc5MjQ5MDA1NzczNUAxNDAxNzU3NDY2MzI4https://www.researchgate.net/publication/6456944_P_Bodies_and_the_Control_of_mRNA_Translation_and_Degradation_Mol_Cell?el=1_x_8&enrichId=rgreq-3066aad4-419b-48b3-a8da-2929dbaaf062&enrichSource=Y292ZXJQYWdlOzIzOTYwNjg3O0FTOjEwMzc5MjQ5MDA1NzczNUAxNDAxNzU3NDY2MzI4

7/25/2019 1645_ftp

4/16

Rsp5p controls gene expression at the entry of stationary phase 3

Table 1.Yeast strains used in this work

Strain Description Origin

BY4742 MAT, his3-1, leu2-0, lys2-0, ura3-0 EUROSCARF

BY4742spi1 BY4742spi1::KanMX4 EUROSCARF

YPH499 MATa,his3-200, leu2-1, lys2-801, trp1-1, ade2-101, ura3-52 F. Abe

FAY18A YPH499 HPG1-1(Pro514>Thr) F. Abe

FAY29E YPH499 HPG1-4(Ala799>Thr) F. Abe

FAJ72 YPH499 bul1::URA3,bul2::HIS3 F. Abe

yYDcG YPH499 DCP2-GFP(kanMX) This work

yHDcG FAY18ADCP2-GFP(kanMX) This work

yBDcG FAJ72DCP2-GFP(kanMX) This work

yYDhG YPH499 DHH1-GFP(kanMX) This work

yHDhG FAY18ADHH1-GFP(kanMX) This work

yBDhG FAJ72DHH1-GFP(kanMX) This work

BY4741 MATa,his3-1, leu2-0, met17-0, ura3-0 EUROSCARF

BY4741ubi4 BY4741ubi4::KanMX4 EUROSCARF

BY4741ubc7 BY4741ubc7::KanMX4 EUROSCARF

BY4741ubp4 BY4741ubp4::KanMX4 EUROSCARF



BY4741ubc2 BY4741ubc2::KanMX4 EUROSCARFBY4741ubc1 BY4741ubc1:: KanMX4 EUROSCARF

BY4741ubc4 BY4741ubc4:: KanMX4 EUROSCARF

BY4741ubc5 BY4741ubc5:: KanMX4 EUROSCARF

W303-1a MATaade2-1, ura3-1, leu2-3, his3-1, trp1-1 R. Rothstein

2344c (WT) MAT ura3 B. Andr e

27038a (npi1) MAT npi1 ura3 B. Andr e

OS2718(bul1bul2) MAT bul1::KanMX4bul2::KanMX4 ura3 B. Andr e

MLY40 (WT) MAT ura3-52 J. Heitman

XPY80(sok2) MAT sok2::hygB ura3-52 J. Heitman

stationary phase and that it has a positive role in p-

body formation in the glucose-depleted condition.

Materials and methods

Yeast strains and growth conditions

The yeast strains used in this work are listedin Table 1. For yeast growth, YPD medium (1%w/v yeast extract, 2% w/v bactopeptone, 2% w/vglucose), SD or SC medium (0.17% w/v yeastnitrogen base without amino acids and ammoniumsulphate, 0.5% w/v ammonium sulphate, 2% w/vglucose) supplemented with the required aminoacids, were used. Cultures were incubated at 30 Cwith shaking. Solid plates contained 2% agarand the specific plates contained 0.008% SDS,0.1 g/ml rapamycin and cycloheximide.

To analyse stress resistance, the cells from expo-nential cultures in the YPD medium were affectedby the following adverse conditions: ethanol addi-tion up to 12% v/v final concentration and incu-bation for 1 h; heat shock at 45 C for 1 h; shift

to YPD containing 1 M KCl and incubation for

1 h (osmotic stress); and YPD buffered at pH 2or 10 for acid or basic stress, respectively. In allcases, cell viability was determined by counting thenumber of colonies growing in YPD plates fromappropriate dilutions of cultures. To analyse resis-tance to oxidative stress, the diameter of the growthinhibition region produced by a paper disc with10 l 33% H2O2 on stationary phase cultures onYPD plates was measured (Stephen et al., 1995).

For microscopy studies, the cells in the exponen-tial phase were transferred from YPD to YP for10 min and were then washed with SC. Observa-

tions were made using a Nikon PCM 2000 micro-scope, using a 100 objective with a 3 zoom.

Construction of yeast strains

In order to analyse the transcriptional regulation ofthe SPI1 gene, a fusion of the promoter of thisgene with the lacZreporter gene was constructedin the Yep357 plasmid. For this purpose, we clonedthe intergenic region of the SPI1PEA2 intergenic

Copyright 2009 John Wiley & Sons, Ltd. Yeast2009; 26: 1 15.

DOI: 10.1002/yea

7/25/2019 1645_ftp

5/16


region by PCR and added the XbaI and EcoRIrestriction sites to the oligonucleotides used (seeSupporting information, Table S1).

Dcp2p and Dhh1p proteins were C-terminaltagged with GFP from plasmid pFA6GFP(S65T)KanMX6, following the PCR-based gene modifica-

tion method described by Longtine et al. (1998),using the appropriate oligonulceotides (see Sup-porting information, Table S1). The appropriatestrains were transformed.

-galactosidase analysis

TheSPI1placZfusion expression was determinedas -galactosidase activity via the method of per-meabilized cells, using ONPG as a substrate, asdescribed by Adams et al. (1997). To detect bluecolonies, plates containing SC ura + Xgal

0.4 mg/ml + phosphate buffer, pH 7, were used.The white colonies defective inSPI1placZ fusionactivity were transformed with a library constructedin the episomal plasmid YEp32 (a gift from J. C.Igual). The transformation was replica-plated on X-gal plates.

RNA analysis

RNA isolation, quantification and analysis werecarried out as previously described (Carrascoet al.,2003). The expression of the SPI1 gene on several

strains and conditions was followed by Northernblot analysis, using a specific probe for this geneobtained with the oligonucleotides SPI1-F/G (seeSupporting information, Table S1).

For microarray analyses, cDNA preparation,labelling and hybridization were carried out asdescribed by Fazzio et al. (2001). The combi-nations for hybridization Cy3 Cy5 were: WTaHPG11a, HPG11bWTb, where the a and bsamples were obtained from two independent cul-tures of each strain. The intensity obtained ineach channel for each pair of microarrays wasnormalized by Lowess (Yang et al., 2002). Theoverrepresentation of categories containing func-tionally related genes in each strain was statis-tically analysed using the Function Associatetool (http://llama.med.harvard.edu/cgi/func/funcassociate). The analysis of the transcription fac-tors involved in the expression of differentiallyexpressed genes was carried out with the YEAS-TRACT package (http://www.yeastracts.com).

The expression of some genes with the differen-tial expression in the wild-type (WT) and HPG1-1strains in the microarray analysis was also analysedby semiquantitative RTPCR. cDNA preparationwas carried out as previously described (Jimenez-Martet al., 2007). For the amplification of specific

genes in the resulting cDNA, it was diluted 20-foldand 5 l were used for a PCR reaction carried out ina final volume of 15 25l, containing the primers(0.5 mMof each), dNTPs (0.2 mMof each), MgCl2(3 mM), buffer and 1 U DNA polymerase Biotaq(Bioline) fromThermus aquaticus YT-1. The reac-tion conditions were: 1 cycle at 94 C for 3 min,20 25 cycles at 94 C for 1 min, 1 min at the opti-mal primer hybridization temperature in each case,1 min at 72 C and, finally, a 10 min cycle at 72 C.The ACT1 gene was used to normalize the data.The specific oligonucleotides used for the analysis

of each gene considered are shown in the Support-ing information.

Western blotting

To prepare protein extracts, 2.5 OD unit cellswere collected by centrifugation and were resus-pended in 100 l water, then 100 l NaOH 0.2M were added and the preparation was incubatedfor 5 min. After centrifuging at 12 000 r.p.m.for 1 min, the pellet was resuspended in 50 l 2loading buffer (TrisHCl 150 mM, pH 6.8, DTT

300 mM, SDS 6%, bromophenol blue 0.3%, glyc-erol 30%) and incubated at 95 C for 5 min. Aftercooling and centrifuging (3000 r.p.m. for 10 min),the supernatant was collected. The total proteinconcentration was determined by the Bradfordmethod (BioRad), and 50 g protein was used foreach analysis. After SDS PAGE electrophoresisin 7.5% polyacrylamide, proteins were transferredto a 0.45 m nitrocellulose membrane (Trans-Blot,1620113, Bio-Rad) by electrotransference. Immun-odetection was carried out using a GFP antibody(Sigma). This primary antibody and the secondaryantibody (peroxidase anti-rabbit) were diluted at1/10 000 and 1/50 000, respectively. Blocking andincubation with the antibodies were carried outin TBST buffer (20 mM Tris HCl, pH 8, 0.5 MNaCl, 0.05% v/v Tween 20) with 5% w/v non-fatdried milk. Washes were done in TBST. ECF plusthe Western Blotting detection system (RPN2132,Amersham) were used for detection, following themanufacturers instructions.


DOI: 10.1002/yea
https://www.researchgate.net/publication/11038288_Yang_IV_et_al_Within_the_fold_assessing_differential_expression_measures_and_reproducibility_in_microarray_assays_Genome_Biol_3_0062?el=1_x_8&enrichId=rgreq-3066aad4-419b-48b3-a8da-2929dbaaf062&enrichSource=Y292ZXJQYWdlOzIzOTYwNjg3O0FTOjEwMzc5MjQ5MDA1NzczNUAxNDAxNzU3NDY2MzI4https://www.researchgate.net/publication/11038288_Yang_IV_et_al_Within_the_fold_assessing_differential_expression_measures_and_reproducibility_in_microarray_assays_Genome_Biol_3_0062?el=1_x_8&enrichId=rgreq-3066aad4-419b-48b3-a8da-2929dbaaf062&enrichSource=Y292ZXJQYWdlOzIzOTYwNjg3O0FTOjEwMzc5MjQ5MDA1NzczNUAxNDAxNzU3NDY2MzI4https://www.researchgate.net/publication/11038288_Yang_IV_et_al_Within_the_fold_assessing_differential_expression_measures_and_reproducibility_in_microarray_assays_Genome_Biol_3_0062?el=1_x_8&enrichId=rgreq-3066aad4-419b-48b3-a8da-2929dbaaf062&enrichSource=Y292ZXJQYWdlOzIzOTYwNjg3O0FTOjEwMzc5MjQ5MDA1NzczNUAxNDAxNzU3NDY2MzI4https://www.researchgate.net/publication/11038288_Yang_IV_et_al_Within_the_fold_assessing_differential_expression_measures_and_reproducibility_in_microarray_assays_Genome_Biol_3_0062?el=1_x_8&enrichId=rgreq-3066aad4-419b-48b3-a8da-2929dbaaf062&enrichSource=Y292ZXJQYWdlOzIzOTYwNjg3O0FTOjEwMzc5MjQ5MDA1NzczNUAxNDAxNzU3NDY2MzI4

7/25/2019 1645_ftp

6/16


Results

Spi1p is involved in stress tolerance and isexpressed in the late growth phases

We have previously used the SPI1 promoter as

a biotechnological tool to increase stress toler-ance by overproducing the general stress responsetranscription factor Msn2p (Cardona et al., 2007).We aim now to obtain a better understandingof not only its role in stress resistance but alsoits regulation. First, we tested the effect of thespi1 deletion on viability after exposure to sev-eral adverse conditions. Table 2 shows the viabilityof the wild-type strain and the spi1 mutant afterheat shock, ethanol stress and high and low pH.The strain carrying the deletion is hypersensitiveto heat shock and alkaline stress, while ethanoland acidic stress determine minor, but clear, differ-ences between the wt and mutant strains. There isno influence of the spi1deletion regarding viabil-ity under other stress conditions, e.g. acetaldehydeor osmotic stress (data not shown). Oxidative stresswas measured as the diameter of the halo of inhi-bition caused by hydrogen peroxide. The mutantstrain also shows increased sensitivity to oxida-tive insult (Table 2). To assess the influence ofSpi1p in the late phases of growth, the survivalviability of a culture of the deletion strain in YPD

was measured. The number of viable cells in thespi1 deletion mutant compared to the wild-typewas lower during the postdiauxic and early station-ary phases (Figure 1). Therefore, Spi1p is a proteinthat is relevant to many stress conditions, includingstarvation.

In order to study the expression of the SPI1gene, we constructed a SPI1 promoter-controlledversion of the lacZ reporter gene on a plasmid.To test this reporter gene, we measured the -galactosidase activity along the growth curve in aminimal medium on strain YPH499 (Figure 2A).

The results indicated that the maximal activity of

0

20

40

60

80

100

120

140

160

0 5 10 15 20 25 30 35 40 45

t(days)

N/No

BY4742spi1

Figure 1.SPI1deletion causes decreased cell viability alongwith growth. Exponentially growing cells of wt (BY4742)and spi1 deletion mutant on YPD at OD600 = 0.3 weretaken as the starting point. Samples of cells were taken atdifferent times, diluted and plated on YPD plates to obtainthe cfu number (N), which is expressed as the ratio to theinitial cfu value (N0)

this gene during growth is around 7 h after the cul-ture started, with a dilution of an overnight cultureat OD600 = 0.5. Similar results were observed inthe rich medium YPD using microarrays (Gaschet al., 2000), where the maximum of expression isreached between 612 h (depending on individualexperiments) from a starting point of OD = 0.3,and its expression remains high throughout thestationary phase. However, in our -galactosidaseassay, the enzyme activity decreases at later stagesof growth, probably reflecting the general decreasein translation that happens in the stationary phase

(Gray et al., 2004). Therefore, the SPI1placZconstruct is a useful tool to study the gene expres-sion changes previous to the entry into the station-ary phase.

Expression of theSPI1gene is affected by RSP5

We used the SPI1placZ fusion to identify thegenes involved in SPI1 gene regulation. We car-ried out an EMS mutagenesis and isolated coloniesthat were white on SD plates with low sugar con-centration (0.5% glucose) and X-gal, instead of

the wild-type blue colour. One of those white

Table 2. Stress tolerance in the wild-type and spi1 mutant. The percentage of colony-forming units after the stresscondition is shown

Ethanol Heat shock Low pH High pH Oxidative (mm)

wt 22 1.5% 0.88 0.001% 67 7% 25 2% 3.08 0.15

spi1 17 1.2% 0.42 0.0001% 56 2% 10 1% 3.9 0.3

For oxidative stress, the diameter (mm) of the inhibition halo is shown. All experiments were carried out in triplicate. SD is shown.


DOI: 10.1002/yea

7/25/2019 1645_ftp

7/16


m4 pTRP1 pTAT2B

C

A

0

5

10

15

20

25

30

35

0 8 12 16 20 24

-ga

lactosidase

(a.u

)HPG1-1

time (hours)

WT

4

HPG1-1

E

YPH499

E

rRNA

SPI1

PD PD

Figure 2. SPI1 expression depends on the growth phaselevels and Rsp5p. (A) The SPI1 expression measured as-galactosidase activity, wild-type (YPH499 strain) andthe rsp5 mutant (HPG1-1) throughout its growth inminimal medium using the SPI1 promoterlacZ fusion.-Galactosidase activity was expressed as arbitrary units(a.u.) according to Adams et al. (1997). The experimentswere carried out by triplicate and standard deviation (SD)

is shown. (B) -Galactosidase assays on plates with lowglucose (0.5%) in a mutant unable to express the SPI1placZfusion, transformed with either an empty plasmid (m4) or aplasmid containing genes implied in tryptophan metabolism(pTRP1) or transport (pTAT2). (C)SPI1 mRNA levels inboth wild-type andHPG1-1mutant in either the exponential(E) or postdiauxic (PD) phase

mutants, named m4 (Figure 2B), was transformedwith a multicopy library in order to isolate reg-ulators of SPI1 expression. We found two kindsof plasmids that cause a reversion to the bluecolour (Figure 2B). One contains the high-affinitytryptophan transporter TAT2, and the other con-tains the TRP1 gene that codes for phosphori-bosylanthranilate isomerase, which catalyses thethird step in tryptophan biosynthesis. The strainused in the screening, W303-1a, is a trp1-1 strain.We failed to fully complement the m4 muta-tion with a centromeric plasmid containing neitherthe TRP1 marker nor the TAT2 gene (data notshown). So far, we have been unable to identify

the location of mutation m4, but it seems clear thattryptophan metabolism is apparently important forSPI1 expression.

It is known that the activity of the tryptophanpermeases Tat1p and Tat2p is controlled by theubiquitin ligase Rsp5p (Abe and Iida, 2003) and

that Rsp5p also controls the stress response (Hai-taniet al., 2006). Therefore, we tested the role of amutant of the RSP5 gene calledHPG1-1 (Abe andIda, 2003) in SPI1lacZ fusion (Figure 2A). Themutant had lower levels of -galactosidase activ-ity at the entry of the stationary phase, indicating apositive role of Rsp5p in SPI1 expression at thelate phases of cell growth. To confirm that thiseffect is indeed located at the transcriptional level,a Northern blot analysis of SPI1 was carried outusing the same mutant (Figure 2C), and showed adefect in the transcriptional induction of SPI1 in

the postdiauxic phase.

Role of the ubiquitination machinery inSPI1expression

We next attempted to gain a better understandingof the role that ubiquitination plays in SPI1 geneexpression, using different mutations in the genesrelated to this process. RSP5 is an essential geneand HPG1-1 is one of the mutants isolated fromthis gene. In this case, it carries a mutation ofthe Pro 514 to Thr in the catalytic centre of the

enzyme, which causes a semi-dominant mutationthat confers resistance to high pressures, given itsinability to ubiquitinate and to lower the levels oftryptophan permease Tat2p (Abe and Ida, 2003).To test whether other mutations in the RSP5gene show the same phenotype, we transformedone called npi1, which plays a clear role in thedegradation of the amino acid transporters Gap1pand Fur4p (Heinet al., 1995) with the SPI1placZfusion. A decreased expression was also observedalong the culture curve (Figure 3A). Bul1p andBul2p are two functional homologous proteins thatinteract with Rsp5p and are sometimes referred toas E4 proteins (Hoppe, 2005). The deletion of bothgenes also leads to a reduction ofSPI1 expressionat the entry of the stationary phase (Figure 3B),which also indicates a positive contribution of thesetwo proteins to SPI1 expression.

We then tested the effect of deleting other com-ponents from the ubiquitination machinery, usinga single measurement of -galactosidase activity


DOI: 10.1002/yea

7/25/2019 1645_ftp

8/16


A

B

0

0.5

1

1.5

2

WT

ubi4

ubc1

ubc2

ubc4

ubc5

ubc7

ubp4

ubp6

ubp14

sok2

C

-galactosidase

(a.u.)

0

5

10

15

20

25

30

0 4 8 12 16 20 24

-gal

actosidase

(a.u.)

time (hours)

-galactosidase

(a.u.)

time (hours)

05

10

15

20

25

30

35

40

0 8 12 16 20 24

WTbul1bul2

WT

npi1

4

Figure 3. The ubiquitination machinery controls the SPI1expression. (A) -galactosidase levels of the SPI1lacZfusion expression of 2344c strain (WT) and 27038a npi1mutant strain throughout its growth in SC-URA medium.(B) Same as (A) for TB50a (WT) and JC60-4b (bul1bul2)strains. (C) The SPI1lacZ fusion expression in several

mutants of the ubiquitination pathway after 8 h of growthfrom an OD = 0.3 starting point. In this case the activity isreferred to as wt activity = 1. All experiments were carriedout in triplicate and SD is shown

in the postdiauxic phase (Figure 3C). S. cere-visiae ubiquitin is a 76-amino acid protein encodedby four structural genes, UBI1, UBI2, UBI3 andUBI4. While each of these genes is reported tobe expressed during yeast exponential growth, only

UBI4 is shown to be strongly inducible by stressessuch as starvation, heat and DNA-damaging agents(Simon et al., 1999). However, UBI4 deletion hasno effect on SPI1 expression (Figure 3C). There-fore, this stress-regulated version of ubiquitin is notessential to the regulation of the stress-responsive

gene SPI1. There are around 13 genes whichencode for the ubiquitin-conjugating (E2 or UBC)enzymes in yeast (Hiraishi et al., 2006). Ubc4pand Ubc5p E2s have been named stress ubiquitin-conjugating enzymes because of their role in stressresistance (Arnason and Ellison, 1994). Howevertheir deletion has no effect onSPI1 expression. Thesame result was obtained with the UBC2/RAD6mutant, an E2 involved in the N-end rule degra-dation (Dohmen et al., 1991). UBC1 deletion hasa negative effect, as it decreases lac Z activity, so itis apparently necessary to achieve fullSPI1 expres-

sion. Ubc1p acts with Rsp5p to signal Gal2p for aneffective internalization by endocytosis and subse-quent proteolysis in the vacuole (Horak and Wolf,2001). Ubc7p physically and genetically interactswith Rsp5p to achieve proper chromatin formation,and it has been proposed that it is a bona fide E2for Rsp5p (Arnason et al., 2005). UBC7 deletionshows an increase ofSPI1 expression (Figure 3C),which either indicates that it may compete withother E2 enzymes, such as Ubc1p for Rsp5p, or actson an opposite pathway, maybe at the chromatinlevel, causing a more relaxed chromatin on the

SPI1 promoter. Therefore, different E2 enzymescould regulate Rsp5p activity in order to properlycontrol its function as a gene expression regulator.

On the other hand, there are 17 ubiquitin-specificprotease genes (UBP) in yeast. Ubp4p (Doa4p),Ubp6p and Ubp14p are involved in the degrada-tion of Tat2p mediated by Rsp5p (Miura and Abe,2004). We tested the effect of the deletion of thesegenes in SPI1 expression (Figure 3C). The dele-tion of UBP4 did not decrease the SPI1placZlevels and even had an slightly positive effect.Nonetheless, the deletion of both UBP6 andUBP14 dramatically decreased theSPI1lacZ lev-els (Figure 3C), indicating that these proteins playan important role in this particular event of thegene expression, by triggering the degradation ofan unknown factor that probably acts negativelyon SPI1 expression. In conclusion, the ubiquitina-tion machinery shows a complex pattern in termsof theSPI1 expression during starvation, with bothpositive and negative regulatory effects.


DOI: 10.1002/yea

7/25/2019 1645_ftp

9/16


Transcriptomic analysis of thersp5mutantindicates an induction of the genes involved inprotein biosynthesis

In order to understand the role of Rsp5p in yeastgene expression at the beginning of the stationary

phase, a transcriptomic analysis was carried outwith the mutant HPG1-1 and the correspondingwild-type (YPH499) strain by microarrays. For thispurpose samples were obtained from both strains atthe entry of the stationary phase after 8 h growthin YPD from a starting point of OD600 = 0.3.It is worth mentioning that no differences in thegrowth phase of the culture were found under theconditions used (data not shown).

In theHPG1-1 mutant, the expression of 88 non-dubious genes was induced by a factor of 3, 118genes by a factor of 2 and 255 genes by a fac-

tor of 1.5. Among the repressed genes, 14 wererepressed by a factor of 3, 54 by a factor of 2and 192 by 1.5. We used the Funcassociate toolto detect the functional categories overrepresentedamong the genes that had a differential expressionof 2 or more. Among the genes induced (Table 3A),most belonged to the overlapping categories relatedto translation (97/118 genes; p value = 5.6e76),particularly to the cytosolic ribosome (96/118;p value = 3.6e144). Some of these proteins werealso involved in other events, such as ribosomeassembly (16 genes) and telomere maintenance (18

genes). Of the 22 genes not related to the ribosome,the most significant category was the cell wallcomponents (6/22;p value = 1.5e6). The Yeastracttool, which groups genes by the transcriptional fac-tor regulating them (Teixeira et al. 2006), showedthat 83.9% of these genes were controlled by theRap1p transcription factor, while 80.5% were con-trolled by Ifh1p, two well-known regulators of ribo-some biogenesis (Wade et al., 2004).

The categories among the genes repressed inthe HPG1-1 mutant are more diverse but are allrelated to metabolic processes, such as carboxylicacid metabolism (13/54; p value = 1.16e-06), theamino acid metabolic process (10 genes; p value =5e-06) and proline transporters (3 genes;p value =2.2e-06) (Table 3B). Using the Yeastract tool,61.1% of the genes were controlled by the cellcycle regulator Sok2p (although the SOK2 mRNAitself is not changed in the HPG1 mutant). SPI1was also downregulated in the microarray analy-sis, together with a few stress-response genes. To

Table 3. Genes with a differential expression in HPG1-1mutant vs. the wild-type strain

Categories Genes

(A) Induced

Translation RPL9A(8.19),RPS5(7.42),RPS22(7.35),

RPL32(6.82),RPL24A(6.71),RPS15(6.56),RPL8B(6.46),RPS4B(6,19),RPS0A(6.15),

RPS9B(6.13),RPS9A(6.12),RPS24B(6.12),

RPS1B(5.9),RPL22A(5.84),RPS24A(5.77),

RPL8A(5.75),RPP2A(5.7),RPL37A(5.65),

RPS4A(5.63),RPS16B(5.52),RPL6A(5.45),

RPS17B(5.4),RPL25(5.34),RPP0(5.34),

RPS17A(5.33),RPL9B(5.32),RPL12A(5.27),

RPL26B(5.24),RPS10A(5.23),RPL6B(5.23),

RPL42A(5.19),RPL13B(5.11),RPS1B(5.11),

RPS26B(5.06),RPL20A(5.02),RPL19A

(4.98),RPL5(4.95),RPS6A(4.94),RPS31

(4,90),RPL12B(4.79),RPL21A(4.76),RPS1A

(4.76),RPL21B(4.75),RPL42B(4.72),RPL2A

(4.70),RPS13(4.63),RPL20B(4.62),RPL34A(4.54),RPS14A(4.53),RPL19B(4.49),

RPL43A(4.48),RPS3(4.47),RPS2(4.44),

RPL39(4.43),RPS12(4.42),RPS21B(4.33),

RPL23A(4.29),RPL27B(4.20),RPL13A

(4.17),RPL34B(4.15),RPL33A(4.15),RPL30

(4.14),RPL33B(4.06),RPS27B(4.03),RPL29

(4.03),RPS14B(3.97),RPL15A(3.81),

RPL36B(3.78),RPS29B(3.71),RPS29A

(3.67),RPL35B(3.62),RPL15B(3.56),

RPL43B(3.57),RPL37B(3.57),RPL36A

(3.55),RPL1A(3.50),RPL35A(3.48),RPS25B

(3.44),RPL11B(3.39),RPS20(3.37),RPL38

(3.23),RPL11A(3.22),RPS25A(3.18),RPL1B

(3.13),RPL22B(3.13),RPP2B(2.91),STM1

(2.88),RPL4B(2.70),RPS30A(2.62),RPL41A

(2.55),RPS30B(2.54),RPL41B(2.47),

RPS28B(2.41),RPP1A(2.33),RPL4A(2.32),

TEF1(2.21),RPS28A (2.12)

Cell wall GAS3(3.05),YLR042c(3.00),PLB2(2.65),

SCW10(2.33),SRL1(2.13),MCD4(2.00)

Other CHA1(19.62),RNR1(2.74),NMR1(2.67),

POL30(2.57),BTN2(2.48),MIS1(2.34),

HXK1(2.30),ACM1(2.25),ARO10(2.18),

RNH203(2.15),SCP160(2.10), TOS4

(2.08),SPO16(2.08),PMI40(2.04),GND1

(2.02)

(B) RepressedAmino acid

metabolism

SER3(3.11),AGX1(3.09),MET3(2.90),

YAT2(2.70),LEU2(2.57),MET17(2.53),

IDP2(2.11),GDH1(2.11),MET2(2.10),

MLS1(2.07)

Proline transport GAP1(3.19),PUT4(3.10),AGP1(2.25)

Stress HSP12(11.94),HSP26(4.11),MEI5(2.55),

GRE1(2.48),SPL2(2.40),DDR2(2.29),SPI1

(2.25),TSA2(2.25),NCE103(2.13)


DOI: 10.1002/yea

7/25/2019 1645_ftp

10/16


Table 3.Continued

Categories Genes

Other PHO89(4.73),FMP45(4.36),FMP16(4.11),

NDE2(3.73),NQM1(3.63),SPS100, (3.57),

PHM6(2.95),YCT1(2.93),GND2(2.88),

MSC1(2.78),AQY1(2.71),PUR5(2.67),NCE102(2.60),FRM2(2.58),GMP2(2.40),

LAP4(2.30),BOP2(2.26),PHM7(2.20),

PDH1(2.17),ALD4(2.15),FLR1(2.06),

ZRT1(2.04),RMD6(2.03)

Unknown YMR107w(6.93),YDL218w(5.04),

YKL071w(2.82),YHR033w(2.60),

YHR140w(2.48),YBR285w(2.32),YAL061w

(2.24),YGR154c(2.16),YDL223c(2.10)

Those genes that are induced or repressed by>2 are shown, and

their induction or repression values are shown in brackets.

test whether Sok2p is a positive regulator ofSPI1expression, we transformed a sok2 deletion strainwith the SPI1placZ fusion. We found that Sok2pis indeed necessary to fully activate the fusionunder starvation conditions (Figure 3C).

To confirm the array data, a semi-quantitativeRT PCR analysis of selected genes was carriedout, using the actin gene ACT1 as a control(Figure 4). RPL9 was used as a representative ofthe ribosomal protein cluster. It was clearly overex-pressed in the mutant strain.CHA1, a gene involvedin serine catabolism, was the most overexpressedgene in the HPG1-1 mutant, according to the arraydata (19.6-fold; see Table 3). Its induction was con-firmed by RT PCR. GAS3 encodes for a 1,3--glucanosyltransferase representative of the cell wallcluster that also showed the expected transcriptionpattern. Among the repressed genes, examples ofeach functional family were tested, and the repres-sions of SPI1 itself, the stress-responsive gene

HSP26, the amino acid biosynthetic genes SER3and MET3 and the amino acid permease GAP1were also confirmed (Figure 4).

Stress resistance inHPG1and bul1/2mutants

We went on to attempt to relate the expression datato the role ofRSP5 mutants and bul1/2 deletion instress resistance. In addition to theHPG1-1 mutant,we used a different allele ofRSP5, HPG1-4, whosemutation is placed in a different region of theRSP5-coding region, providing a thermosensitivephenotype (Abe and Ida, 2003). Some cell wall-related genes were induced in the HPG1-1 mutant(Table 3A), a fact that may indicate an imbalancein cell wall assembly or a response to cell walldamage. We grew all these mutants on platescontaining 0.008% SDS to detect a cell wall defect.

Indeed, we found this to be the case, particularlyfor the HPG1-1 and bul1/2 mutants (Figure 5A).

The identification of genes involved in aminoacid metabolism in our microarray analysis sug-gests a putative involvement of Rsp5p in theresponse to nutrient limitation. To address this pos-sibility, our mutant cells were grown in the pres-ence of rapamycin, which inhibits the conserved

ACT1

SPI1

HSP26

SER3

MET3

GAP1

RPL9a

GAS3

CHA1

WT

HPG1-1

Figure 4. Gene expresson analysis by semi-quantitativeRTPCR of several genes genes that were either identifiedas being induced or repressed in the HPG1-1mutant at thepost-diauxic shift.ACT1was used as a control

Figure 5.Effects of toxic agents on the growth of ubiquitination mutants. TheHPG1-1, HPG1-4and bul1bul2mutantswere replica-plated on (A) SDS 0.008% or rapamycin 0.1 g/ml and (B) cycloheximide 0.1g/ml


DOI: 10.1002/yea

7/25/2019 1645_ftp

11/16


WT Dcp2p-GFP HPG1-1 Dcp2p-GFP bul1bul2Dcp2p-GFP

1M KCl 15

YP 15

A

Dhh1p-GFP

Dcp2p-GFP

Ponceau

Ponceau

KCl 1M / 15 YP / 15

WT

HPG1-1

bul1bul2

WT

HPG1-1

bul1bul2

C

B WT Dhh1p-GFP HPG1-1 Dhh1p-GFP bul1bul2Dhh1p-GFP

1M KCl 15

YP 15

Figure 6.TheRSP5mutant is defective in p-body formation. Microscopy after 15 min without glucose (YP) or with osmoticstress (1 M KCl) in WT, HPG1-1 and bul1bul2 strains transformed with (A) Dcp2pGFP fusion and (B) Dhh1pGFPfusion. (C) Western blot of the Dcp2pGFP and Dhh1pGFP protein levels of these strains under the same stressconditions, using an anti-GFP antibody


DOI: 10.1002/yea

7/25/2019 1645_ftp

12/16


protein kinase that links nutrient status and cell pro-liferation, target of rapamycin (TOR). Our muta-tions were hypersensitive to rapamycin, indicatingthat ubiquitination machinery contributes to theresponse to nutrient starvation.

A relationship between Rsp5p and translation

accuracy has been recently reported (Kwapiszet al., 2006), according to which a S. cerevisiaestrain carrying an rsp5-13 mutation shows alteredsensitivity to antibiotics and a slower rate of trans-lation. The results obtained in the microarray andin the semi-quantitative RT PCR analysis indi-cate that there is a higher level of transcriptsrelated to protein biosynthesis in the mutant HPG1-1 under stationary phase conditions. In order toconfirm this effect on the translation of the rsp5mutant alleles that we used and the role of theBUL1/2 role in translation, experiments of resis-

tance to the translation inhibitor cycloheximidewere carried out (Figure 5B). The HPG1-1 allelepresented a significant defect on translation. How-ever, the HPG1-4 allele did not have such a sig-nificant growth defect. Therefore, the cyclohex-imide sensitivity showed some allele specificity.The bul1/2 deletion also revealed an increasedcycloheximide sensitivity. These data confirm therole of Rsp5p Bul1p Bul2p in translation andindicate that this role may be related to the alter-ation of the translation machinery expression.

RSP5is involved in p-body formation understress conditions

According to our microarray data an increasedexpression of the ribosomal proteins was foundin the rsp5 mutant. To a lower extent, there wasalso an increase in the levels of several factorsinvolved in translation elongation, such as the EF-1 component TEF1 (2.2-fold induction), EF-1component EFB1 (1.87-fold), EF-2 componentsEFT2 (1.96-fold) and EFT1 (1.62), and the EF-3componentYEF3 (1.77-fold). In higher eukaryotes,ribosomal and translational elongation factors areco-regulated at the translation initiation level ina TOR-dependent manner during nutrient depriva-tion, given the presence of a 5-terminal oligopy-rimidine tract (TOP) at their 5-UTR (Hamiltonet al., 2006). This regulation system is not presentinS. cerevisiae, but the co-regulation of these genesin theHPG1-1 mutant suggests that a common reg-ulation mechanism may be involved in the expres-sion of this family of functionally related genes.

Rsp5p plays a role in gene expression at the post-transcriptional level (see Introduction). Thereforewe tested the effect of our ubiquitination mutants inanother post-transcriptional event related to stressand nutrient starvation, that of the formation of pro-cessing bodies (p-bodies).

To analyse this event, we fused the green flu-orescent protein GFP to two proteins implicatedin mRNA degradation that are well established p-body markers, Dcp2p and Dhh1p in the HPG1-1and the bul1/bul2 mutants. Under glucose depri-vation stress (15 min in YP) and osmotic shock(15 min in 1 M KCl), the p-bodies were clearlyformed in the wild-type cells, both when Dcp2p(Figure 6A) and Dhh1p (Figure 6B) were fused toGFP. In theRSP5 mutant tested,HPG1-1, the num-ber and intensity of the p-bodies decreased for bothGFP fusions (Figure 6) in both conditions, particu-

larly in the starvation condition. This effect was notseen in the bul1/bul2 mutant, where there is stilla high number of cells containing p-bodies afterthe stress. Therefore, the Rsp5p has an importantrole in the formation of p-bodies under differentstress conditions without the help of Bul1/2p inthis particular event.

In order to rule out a defect in the fusion pro-tein amount, we carried out a Western blot analy-sis using an anti-GFP antibody (Figure 6C). Thelevels of GFP-fused Dcp2p and Dhh1p proteinswere similar in the HPG1-1 mutant compared to

the wild-type. Therefore, Rsp5 seems to affect theassembly of the p-bodies during these environmen-tal changes, not their protein levels.

Discussion

In this work, we have used the SPI1 gene as amarker of the entry into the stationary phase. Spi1pis a GPI-anchored cell wall protein that plays apositive role in stress response. Its deletion causedan increased sensitivity to various insults, such asheat shock, high ethanol concentration, extreme pHvalues, oxidative stress and starvation (Table 2).As a typical stress-regulated gene, SPI1 expres-sion is induced under many adverse conditions(Gaschet al., 2000) and is highly expressed in thelate growth phases under both laboratory (Gaschet al., 2000) and industrial conditions (Puig andPerez-Ortn, 2000). We have constructed a fusionof the SPI1 promoter to lacZ to identify new


DOI: 10.1002/yea

7/25/2019 1645_ftp

13/16


regulators of its nutrient-controlled expression. Itis known that increasing tryptophan concentrationor overexpressing tryptophan transporters in trypto-phan auxotrophs increases tolerance to weak acids(Baueret al., 2003) and to ethanol (Hirasawaet al.,2007). We found that multicopy plasmids contain-

ing the tryptophan biosynthetic gene TRP1 and thehigh-affinity tryptophan transporter TAT2 suppressa mutation that causes lowSPI1 expression. There-fore, the effects of tryptophan metabolism on stressare also applied toSPI1 gene expression. Similarly,we have found that TAT2 overexpression regulatesa similar fusion of another starvation-induced gene,YGP1 (data not shown).

A known link between stress resistance and tryp-tophan transport is the ubiquitin ligase Rsp5p. Itsdeletion leads to sensitivities to various environ-mental challenges and it plays a role in Tat2p

degradation under starvation. We used a strain con-taining the HPG1-1 allele of RSP5 to prove therole of this gene in SPI1 expression (Figure 2).This mutant was isolated by Abe and Ida (2003)as a semi-dominant mutant that allows growth athigh pressures, due to its incapacity to degradeTat2p. We have further studied the contribution ofthe ubiquitination machinery to SPI1 expression(Figure 3). The analysis of the double mutant ofthe Rsp5p interacting proteins Bul1p and Bul2phave established a clear function of this complexin SPI1 gene activation at entry to the station-

ary phase. Of the potential E2 of Rsp5p tested,we found that Ubc1p and Ubc7p have a positiveand negative effect, respectively, on the controlof the SPI1 expression. Therefore, it seems thatUbc1p is necessary for the ubiquitination reac-tion that leads to SPI1 activation, and that Ubc7competes with it, or activates an antagonistic path-way. Ubp6 and Ubp14 may contribute to degrad-ing whatever target may lead to the SPI1 induc-tion, because the deletion of both proteins leads toa defect in SPI1 expression. Therefore, it seemsthat Rsp5p Bul1/2p/Ubc1/Ubp6/14p may degradea factor that negatively affects theSPI1 gene induc-tion at the entry to stationary phase. There is noevidence in the global analysis of Rsp5p-mediatedubiquitination of Spi1p as a direct target of Rsp5pactivity (Kus et al., 2005).

We carried out a global analysis to further studythe role of Rsp5p in gene expression at the onsetof the stationary phase. A small number of genesshow lower levels in the HPG1-1 mutant, together

with SPI1. The presence of the genes involvedin proline transport and amino acid metabolism issignificant, which provides a clue that links Rsp5pto the response to the nutritional status. Sok2ptranscription factor controls 61% of the genesrepressed in the mutant HPG1-1 (Teixeira et al.,

2006). We confirmed that the SPI1lacZ fusionactivation is indeed Sok2p-dependent. SOK2 actsdownstream of a main nutrient control pathway,PKA, to regulate the expression of those genesthat are important for growth and development(Ward et al., 1995). Rsp5p may somehow regulateSok2p activity when the stationary phase begins.There is ongoing controversy as to whether Rsp5pcontrols the activity of the transcription factorGln3p, involved in nitrogen catabolite repression(Crespo et al., 2004; Feller et al., 2006). Ourarrays do not suggest a global control of the

nitrogen catabolite repression regulon by Rsp5punder the conditions tested. It is intriguing thatthe gene which displays the greatest induction inthe HPG1-1 mutant (19.6 fold) is CHA1 (Table 3,Figure 4), a gene involved in serine catabolism,while the genes in the serine biosynthetic pathway,such as SER3 (3.1-fold repression), AGX1 (3.1-fold), MET17 (2.5-fold) and SER33 (1.9-fold)are repressed. This indicates a particular role ofRsp5p in the control of the serine and threoninemetabolism by a mechanism that is unknown todate. The antagonistic regulation of CHA1 and

SER3 depends on the Cha4p transcription factorthat can act as both an activator and repressorin response to serine (Martens et al., 2005). Itis temping to suggest that Rsp5p may somehowregulate serine metabolism via this transcriptionfactor or their interacting proteins, although anindirect effect due to the control of amino acidpermease by ubiquitination cannot be ruled out.

A larger number of genes are repressed by Rsp5pat the entry of the stationary phase. They over-whelmingly belong to the category of ribosomalproteins (RP), along with some translation elon-gation factors (see Table 3). Rsp5p is involved intranslation accuracy and mutations on it cause sen-sitivity to cycloheximide (Zwapisz et al., 2002).Cycloheximide is an antibiotic that targets riboso-mal proteins, which leads to a defect on transla-tion. The rsp5 allele we were using, HPG1-1, isalso cycloheximide-sensitive (Figure 5B), despitethe larger amount of ribosomal protein expression,which either suggests additional effects at the post-


DOI: 10.1002/yea

7/25/2019 1645_ftp

14/16


transcriptional level or an unbalanced situation thatleads to a translation deficiency. The translationaccuracy defect detected in the rsp5-13 allele ispartially suppressed by an additional copy of thetranslation elongation factor eIF1ATEF2 (Zwapiszet al., 2002). Ourrsp5 mutation,HPG1-1, activates

the levels of the other coding gene for eIF1A, TEF1(Table 3), and also other transcription elongationfactors, thus reinforcing the role of Rsp5p in trans-lation accuracy. Rsp5p has an antagonistic effecton the TOR pathway at the RP expression level.TOR stimulates RP transcription when nutrients areabundant (Powers, 2004), while Rsp5p repressesRP levels when nutrients become scarce. A sim-ilar antagonistic role between Rsp5p and TORregarding amino acid transporter levels has beenproposed. For instance, under starvation and uponRsp5p/Bul11/2p-dependent ubiquitination, amino

acid transporters are internalized, transported to thevacuole and degraded by vacuolar hydrolases (Abeand Ida, 2003). TOR inhibits this turnover whennutrients are high in the medium via Npr1p kinase(Schmidtet al., 1998). A similar molecular mech-anism has been proposed to explain the role ofRsp5p in the regulation of the nitrogen metabolismcontroller Gln3p (Crespo et al., 2004). TOR acti-vates Npr1p kinase when nutrients are high, and inturn inhibits Rsp5p activity. Under nitrogen limi-tation, Rsp5p would be active and would stimulatethe Gln3p shift to the nucleus. However, this effect

seems to be ammonia-specific (Tate et al., 2006).TheHPG1-1 mutant is rapamycin-sensitive, whichindicates that Rsp5p works in the same pathway asTOR, even though they have opposite effects onthe RP levels.

Outside the RP genes, the transcriptomic effectof rsp5 mutation does not match that caused byeither TOR deletion or rapamycin treatment underdifferent conditions (Hardwicket al., 1999; Shamjiet al., 2000; Chen and Powers, 2006). This suggeststhat the Rsp5p mechanism may be either differentor located at another level of the gene expressionother than transcription. We have found a role ofthe ubiquitination machinery on p-body formationfor the very first time. Under several stresses,such as sudden glucose deprivation, proteinRNAaggregates (p-bodies) are formed. It is known thatthe mRNA can exit p-bodies and resume translationwhen glucose is added back. The mutation ofRsp5p results in a decrease of p-body formationunder sudden starvation conditions (see Figure 6).

It has been postulated that the mRNAs in theseaggregates formed during the stationary phase aremore resistant to extraction (Aragon et al., 2006).We postulate that the increase in the mRNA levelsthat we observed in the transcriptomic analysis ofthe HPG1-1 mutants may be related to the fact

that a deficiency in p-body formation increasesthe pool of protein-free mRNA, which is easilyextracted by the usual phenol RNA purification.This would imply that Rsp5p has a specific abilityto sequestrate the mRNAs of RP proteins on p-bodies under starvation conditions. A potential linkbetween ubiquitination and p-body formation maybe the fact that a global screening of Rsp5p targetshas identified the p-body component Dhh1p as atarget of this ubiquitin ligase (the 34th place in themost likely targets; Kus et al., 2005). As a matterof fact, we have observed a slower growth when

Dhh1p is fused to GFP in theHPG1-1 background(data not shown), an observation that may indicatea functional relationship between the two proteins.Additional global studies using proteases to releaseprotein-bound mRNAs would be necessary to fullyassess this hypothesis.

Acknowledgements

We are indebted to F. Estruch for supplying us with the

GFP antibody and J. C. Igual for the multicopy library.

We also thank F. Abe, B. Andre and J. Heitman for the

strains provided. This work was supported by Grant No.AGL2005-00508 from the Ministerio de Educacion y Cien-

cia and Grant No. GRUPOS03/012 from the Generalitat

Valenciana to M.O. and A.A. F.C. was an FPU Fellow of

the Ministerio de Educacion y Ciencia.

Supporting Information

Supporting information may be found in the onlineversion of this article.

References

Abe F, Iida H. 2003. Pressure-induced differential regulation of

the two tryptophan permeases Tat1 and Tat2 by ubiquitin ligase

Rsp5 and its binding proteins, Bul1 and Bul2. Mol Cell Biol23:

75667584.

Adams A, Gottschlings DE, Kaiser CA, Stearns T. 1997. Methods

in Yeast Genetics. Cold Spring Harbor Laboratory Press: Cold

Spring harbor, NY.

Aragon AD, Quinones GA, Thomas EV, et al. 2006. Release of

extraction-resistant mRNA in stationary phase Saccharomyces


DOI: 10.1002/yea

7/25/2019 1645_ftp

15/16


cerevisiaeproduces a massive increase in transcript abundance

in response to stress. Genome Biol 7: R9.

Arnason T, Ellison MJ. 1994. Stress resistance in Saccharomyces

cerevisiae is strongly correlated with assembly of a novel type

of multiubiquitin chain. Mol Cell Biol 14: 78767883.

Arnason TG, Pisclevich MG, Dash MD, et al. 2005. Novel

interaction between Apc5p and Rsp5p in an intracellular

signalling pathway in Saccharomyces cerevisiae. Eukaryot Cell4: 134 146.

Bauer BE, Rossington D, Mollapour M, et al. 2003. Weak organic

acid stress inhibits aromatic amino acid uptake by yeast,

causing a strong influence of amino acid auxotrophies on the

phenotypes of membrane transporter mutants. Eur J Biochem

270: 31893195.

Brengues M, Teixeira D, Parker R. 2005. Movement of eukaryotic

mRNAs between polysomes and cytoplasmic processing bodies.

Science 310: 486489.

Cardona F, Carrasco P, Perez-Ortn JE, et al. 2007. A novel

approach for the improvement of stress resistance in wine yeasts.

Int J Food Microbiol 114: 8391.

Carrasco P, Perez-Ortn JE, del Olmo M. 2003. Arginase activity

is a useful marker of nitrogen limitation during alcoholic

fermentations.System Appl Microbiol 26: 471479.

Chen JCY, Powers T. 2006. Coordinate regulation of multiple and

distinct biosynthetic pathways by TOR and PKA kinases in S.

cerevisiae. Curr Genet49: 281293.

Crespo JL, Helliwell SB, Wiederkehr C, et al. 2004.NPR1 kinase

and RSP5BUL1/2 ubiquitin ligase control GLN3-dependent

transcription in Saccharomyces cerevisiae. J Biol Chem 279:

3751237517.

DeRisi JL, Iyer VR, Brown PO. 1997. Exploring the metabolic

and genetic control of gene expression on a genomic scale.

Science 278: 680686.

Dohmen RJ, Madura K, Bartel B, Varshavsky A. 1991. The N-end

rule is mediated by the UBC2 (RAD6) ubiquitin-conjugating

enzyme.Proc Natl Acad Sci USA 88: 73517355.

Fazzio TG, Kooperberg C, Goldmark JP, et al. 2001. Widespread

collaboration of Isw2 and Sin3-Rpd3 chromatin remodeling

complexes in transcriptional repression. Mol Cell Biol 21:

64506460.

Feller A, Boeckstaens M, Marini AM, et al. 2006. Transduction

of the nitrogen signal activating Gln3-mediated transcription is

independent of Npr1 kinase and Rsp5-Bul1/2 ubiquitin ligase in

Saccharomyces cerevisiae. J Biol Chem 281: 2854628554.

Gasch AP, Spellman PT, Kao CM, et al. 2000. Genomic

expression programs in the response of yeast cells to

environmental changes. Mol Biol Cell 11: 42414257.

Gray JV, Petsko GA, Johnston GC, et al. 2004. Sleeping beauty:

quiescence inSaccharomyces cerevisiae.Microbiol Mol Biol Rev

68: 187206.

Gwizdek C, Hobeika M, Kus B, et al. 2005. The mRNA nuclearexport factor Hpr1 is regulated by Rsp5-mediated ubiquitylation.

J Biol Chem280: 1340113405.

Haitani Y, Shimoi H, Takagi H. 2006. Rsp5 regulates expression

of stress proteins via post-translational modification of Hsf1 and

Msn4 inSaccharomyces cerevisiae.FEBS Lett580: 34333438.

Haitani Y, Takagi H. 2008. Rsp5 is required for the nuclear export

of mRNA of HSF1 and MSN2/4 under stress conditions in

Saccharomyces cerevisiae. Genes Cells 13: 105116.

Hardwick JS, Kuruvilla FG, Tong JK, et al. 1999. Rapamycin-

modulated transcription defines the subset of nutrient-sensitive

signaling pathways directly controlled by the Tor proteins. Proc

Natl Acad Sci USA 96: 1486614870.

Hein C, Springael JY, Volland C, et al. 1995. NPl1, an essential

yeast gene involved in induced degradation of Gap1 and Fur4

permeases, encodes the Rsp5 ubiquitinprotein ligase. Mol

Microbiol18: 7787.

Herman PK. 2002. Stationary phase in yeast. Curr Opin Microbiol

5: 692607.Hicke L. 2001. Protein regulation by monoubiquitin. Nat Rev Mol

Cell Biol 2: 195201.

Hiraishi H, Mochizuki M, Takagi H. 2006. Enhancement of stress

tolerance in Saccharomyces cerevisiae by overexpression of

ubiquitin ligase Rsp5 and ubiquitin-conjugating enzymes.Biosci

Biotechnol Biochem 70: 27622765.

Hirasawa T, Yoshikawa K, Nakakura Y,et al. 2007. Identification

of target genes conferring ethanol stress tolerance to

Saccharomyces cerevisiae based on DNA microarray data

analysis.J Biotechnol 131: 3444.

Hochstrasser M. 1996. Ubiquitin-dependent protein degradation.

Annu Rev Genet30: 405439.

Hohmann S, Mager WH. 2003. Yeast Stress Responses. Springer-

Verlag: Berlin.Horak J. 2003. The role of ubiquitin in down-regulation and

intracellular sorting of membrane proteins: insights from yeast.

Biochim Biophys Acta 1614: 13955.

Horak J, Wolf DH. 2001. Glucose-induced monoubiquitination of

the Saccharomyces cerevisiae galactose transporter is sufficient

to signal its internalisation. J Bacteriol 183: 30833088.

Hoppe T. 2005. Multiubiquitylation by E4 enzymes: one size

doesnt fit all. Trends Biochem Sci 30: 183187.

Hoppe T, Matuschewski K, Rape M, et al. 2000. Activation

of a membrane-bound transcription factor by regulated

ubiquitin/proteasome-dependent processing.Cell102: 577586.

Horie T, Isono K. 2001. Cooperative functions of the

mannoprotein-encoding genes in the biogenesis and mainte-

nance of the cell wall in Saccharomyces cerevisiae. Yeast 18:1493503.

Jimenez-Mart E, Aranda A, Mendes-Ferreira A, et al. 2007. The

nature of nitrogen source added to nitrogen depleted vinifications

conducted by a Saccharomyces cerevisiae strain in synthetic

medium affects gene expression and the levels of several volatile

compounds.Antonie Van Leeuwenhoek 92(61): 75.

Jorgensen P, Rupes I, Sharom JR, et al. 2004. A dynamic

transcriptional network communicates growth potential to

ribosome synthesis and critical cell size. Genes Dev 18:

24912505.

Kaida D, Toh-e A, Kikuchi Y. 2003. Rsp5 Bul1/2 complex is

necessary for the HSE-mediated gene expression in budding

yeast. Biochem Biophys Res Commun 306: 10371041.

Kaminska J, Kwapisz M, Grabilnska K, et al. 2005. Rsp5pubiquitin ligase affects isoprenoid pathway and cell wall

organization in S. cerevisiae. Acta Biochim Pol 52: 207220.

Kus B, Gajadhar A, Stanger K, et al. 2005. A high throughput

screen to identify substrates for the ubiquitin ligase Rsp5. J

Biol Chem33: 2947029478.

Kwapisz M, Cholbinski P, Hopper AK,et al. 2006. Rsp5 ubiquitin

ligase modulates translation accuracy in yeast Saccharomyces

cerevisiae. RNA 11: 17101718.

Longtine MS, McKenzie A III, Demarini DJ, et al. 1998.

Additional modules for versatile and economical PCR-based


DOI: 10.1002/yea

7/25/2019 1645_ftp

16/16


gene deletion and modification in Saccharomyces cerevisiae.

Yeast14: 953961.

Martens JA, Wu PYJ, Winston F. 2005. Regulation of an

intergenic transcript controls adjacent gene transcription in

Saccharomyces cerevisiae. Genes Dev 19: 26952704.

Martin DE, Hall MN. 2005. The expanding TOR signaling

network. Curr Opin Cell Biol 17: 158166.

Max T, Sgaard M, Svejstrup JQ. 2007. Hyperphosphorylation ofthe C-terminal repeat domain of RNA polymerase II facilitates

dissociation of its complex with mediator. J Biol Chem 282:

14113141120.

Miura Y, Abe F. 2004. Multiple ubiquitin-specific protease genes

are involved in degradation of yeast tryptophan permease Tat2

at high pressure. FEMS Microbiol Lett239: 171179.

Neumann S, Petfalski E, Brugger B, et al. 2003. Formation and

nuclear export of tRNA, rRNA and mRNA is regulated by the

ubiquitin ligase Rsp5p. EMBO Rep 4: 11561162.

Parker R, Sheth U. 2007. P bodies and the control of mRNA

translation and degradation. Mol Cell 25: 635646.

Pickart CM. 2004. Back to the future with ubiquitin. Cell 116:

181190.

Powers T. 2004. Ribosome biogenesis: giant steps for a giant

problem.Cell 119: 901902.

Puig S, Perez-Ortn JE. 2000. Stress response and expression

patterns in wine fermentations of yeast genes induced at the

diauxic shift. Yeast16: 139148.

Schmidt A, Beck T, Koller T, et al. 1998. The TOR nutrient

signalling pathway phosphorylates NPR1 and inhibits turnover

of the tryptophan permease. EMBO J17: 69246931.

Simon JR, Treger JM, McEntee K. 1999. Multiple independent

regulatory pathways control UBI4 expression after heat shock

in Saccharomyces cerevisiae. Mol Microbiol 31: 823832.

Sigismund S, Polo S, Di Fiore PP. 2004. Signaling through

monoubiquitination.Curr Top Microbiol Immunol 286: 149185.

Shamji AF, Kuruvilla FG, Schreiber SL. 2000. Partitioning the

transcriptional program induced by rapamycin among the

effectors of the Tor proteins. Curr Biol 10: 15741581.

Somesh BP, Reid J, Liu WF, et al. 2005. Multiple mechanisms

confining RNA polymerase II ubiquitylation to polymerases

undergoing transcriptional arrest. Cell 121: 913923.Stephen DW, Rivers SL, Jamieson DJ. 1995. The role of the YAP1

and YAP2 genes in the regulation of the adaptative oxidative

stress responses of Saccharomyces cerevisiae. Mol Microbiol

16: 415 423.

Tate JJ, Rai R, Cooper TG. 2006. Ammonia-specific regulation of

G1n3 localization inSaccharomyces cerevisiae by protein kinase

Npr1.J Biol Chem 281: 2846028469.

Teixeira MC, Monteiro P, Jain P, et al. 2006. The YEASTRACT

database: a tool for the analysis of transcription regulatory

associations inSaccharomyces cerevisiae.Nucleic Acids Res34:

446451.

Wade JT, Hall DB, Struhl K. 2004. The transcription factor Ifh1

is a key regulator of yeast ribosomal protein. Nature 432:

10541058.

Ward MP, Gimeno CJ, Fink GR, Garrett S. 1995. SOK2 may reg-

ulate cyclic AMP-dependent protein kinase-stimulated growth

and pseudohyphal development by repressing transcription. Mol

Cell Biol 15: 68546863.

Yang IV, Chen E, Hasseman JP, et al. 2002. Within the fold:

assessing differential expression measures and reproducibility in

microarray assays. Genome Biol 3: research 0062.1 0062.12.

Yashiroda H, Kaida D, Toh-e A, Kikuchi Y. 1998. The PY-motif

of Bul1 protein is essential for growth of Saccharomyces

cerevisiae under various stress conditions. Gene 225: 3946.


1645_ftp

Documents