role of skd1 regulators lip5 and ist1-like1 in endosomal ... · role of skd1 regulators lip5 and...

Role of SKD1 Regulators LIP5 and IST1-LIKE1 inEndosomal Sorting and Plant Development1[OPEN]

Rafael Andrade Buono2,3, Julio Paez-Valencia2, Nathan D. Miller, Kaija Goodman, Christoph Spitzer,Edgar P. Spalding, and Marisa S. Otegui*

Department of Botany (R.A.B., J.P.-V., N.D.M., K.G., C.S., E.P.S., M.S.O.), R.M. Bock Laboratories of Cell andMolecular Biology (R.A.B, J.P.-V., K.G., M.S.O.), and Department of Genetics (M.S.O.), University of Wisconsin-Madison, Madison, Wisconsin 53706

ORCID IDs: 0000-0002-6675-3836 (R.A.B.); 0000-0002-6356-0393 (C.S.); 0000-0002-6890-4765 (E.P.S.); 0000-0003-4699-6950 (M.S.O.).

SKD1 is a core component of the mechanism that degrades plasma membrane proteins via the Endosomal Sorting ComplexRequired for Transport (ESCRT) pathway. Its ATPase activity and endosomal recruitment are regulated by the ESCRTcomponents LIP5 and IST1. How LIP5 and IST1 affect ESCRT-mediated endosomal trafficking and development in plantsis not known. Here we use Arabidopsis mutants to demonstrate that LIP5 controls the constitutive degradation of plasmamembrane proteins and the formation of endosomal intraluminal vesicles. Although lip5 mutants were able to polarize theauxin efflux facilitators PIN2 and PIN3, both proteins were mis-sorted to the tonoplast in lip5 root cells. In addition, lip5 root cellsover-accumulated PIN2 at the plasma membrane. Consistently with the trafficking defects of PIN proteins, the lip5 roots showedabnormal gravitropism with an enhanced response within the first 4 h after gravistimulation. LIP5 physically interacts withIST1-LIKE1 (ISTL1), a protein predicted to be the Arabidopsis homolog of yeast IST1. However, we found that Arabidopsiscontains 12 genes coding for predicted IST1-domain containing proteins (ISTL1–12). Within the ISTL1–6 group, ISTL1 showedthe strongest interaction with LIP5, SKD1, and the ESCRT-III-related proteins CHMP1A in yeast two hybrid assays. Through theanalysis of single and double mutants, we found that the synthetic interaction of LIP5 with ISTL1, but not with ISTL2, 3, or 6, isessential for normal plant growth, repression of spontaneous cell death, and post-embryonic lethality.

Secretory and endosomal trafficking pathways con-trol the transport and abundance of proteins through-out the endomembrane system of cells. At the plasmamembrane, proteins such as ion transporters, channels,and receptors are targeted for degradation throughubiquitination followed by endocytic internalization(Barberon et al., 2011; Kasai et al., 2011; Lu et al., 2011;

Shin et al., 2013; Tanno and Komada, 2013; Martinset al., 2015). Internalized plasma membrane proteinsare then delivered in endocytic vesicles to early endo-somes where they can be recycled back to the plasmamembrane or be sorted for degradation in late endo-somes, also called multivesicular bodies (MVBs). AtMVBs, the ubiquitinated plasma membrane cargo pro-teins are sorted into intraluminal vesicles (ILVs) that aredegraded in the vacuolar lumen when mature MVBsfuse with the vacuole (Reyes et al., 2011). If the cargoproteins fail to be sequestered into ILVs, they are mis-sorted to the vacuolar membrane or tonoplast and can-not be degraded efficiently (Babst et al., 2002; Spitzeret al., 2009; Reyes et al., 2011).

The recognition, concentration, and sorting of ubiq-uitinated plasma membrane cargo into ILVs is me-diated by Endosomal Sorting Complex Required forTransport (ESCRT) proteins. In fungi and metazoans,five multimeric ESCRT complexes called ESCRT-0 toESCRT-III and the Vps4p/SKD1-Vta1p/LIP5 complexare involved in endosomal cargo sorting and ILV for-mation. ESCRT-0 binds phosphatidylinositol-3-P andclathrin on the endosomal membranes, recognizes theubiquitinated membrane proteins, and interacts withESCRT-I. Within eukaryotes, only fungi andmetazoanscontain the canonical ESCRT-0 subunits Vps27p/Hrsand Hse-1p/STAM (Leung et al., 2008). Plants andother eukaryotes lacking ESCRT-0 seem to rely on theTOM1 and TOM1-like proteins (Korbei et al., 2013),

1 This work was supported by National Science Foundation grantsno. MCB1157824 to M.S.O., no. IOS1031416 to E.P.S., and funds fromthe Department of Botany to R.A.B.

2 These authors contributed equally to the article.3 Present address: VIB Department of Plant Systems Biology,

UGent, Technologiepark 927, 9052 Ghent, Belgium.* Address correspondence to [email protected] author responsible for distribution of materials integral to the

findings presented in this article in accordance with the policy de-scribed in the Instructions for Authors (www.plantphysiol.org) is:Marisa S. Otegui ([email protected]).

R.A.B., J.P.-V., N.M., K.G., E.P.S., and M.S.O. designed experi-ments; C.S. isolated istl mutant lines; R.A.B. generated plant lines,performed the phylogenetic analysis of ISLT proteins, confocal andelectron microscopy imaging, qPCR, and gravitropism experimentsin E.P.S.’s imaging platform; J.P.-V. performed the yeast-2-hybrid as-says, immunoblot analysis of PIN2-GFP, and histological staining ofistl1 lip5 mutant; N.D.M. and E.P.S. analyzed the data from the grav-itropism experiments; K.G. analyzed plant material; R.A.B., J.P.-V., andM.S.O. wrote the paper with advice from the other authors.

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which are widely distributed in eukaryotes (Hermanet al., 2011) and play the role of an ancestral ESCRT-0module. ESCRT-I and ESCRT-II are also able to bindubiquitin and thought to initiate or stabilize negativecurvature on the endosomal membrane. ESCRT-IIIproteins assemble into filaments with affinity for high-ly curved membranes (Fyfe et al., 2011). ESCRT-IIIproteins do not seem to be able to bind ubiquitin andare found in a closed, inactive state in the cytoplasm,but polymerize in long filaments when recruited byESCRT-II to the endosomal membrane where they con-strict the neck of the nascent ILV and eventually me-diate its release into the MVB lumen (Guizetti andGerlich, 2012; Shen et al., 2014;McCullough et al., 2015).The ESCRT-III complex consists of four core subunitswith essential functions on MVB formation: VacuolarProtein Sorting20/Charged Multivesicular Body Pro-tein6 (Vps20p/CHMP6), Suc Non-Fermenting7 (Snf7p)/CHMP4, Vps24p/CHMP3, and Vps2p/CHMP2, andthree accessory subunits with regulatory functions,Did2p/CHMP1, Vps60p/CHMP5, and Increased SaltTolerance1 (IST1; Babst et al., 2002; Nickerson et al., 2006;Azmi et al., 2008; Nickerson et al., 2010; Henne et al.,2011). The disassembly and recycling of the ESCRT-IIIcomponents back to the cytoplasm and the continu-ous release of ILVs into the endosomal lumen requirethe ATPase mechanozenzyme Vps4p/SKD1, which co-assembles with its cofactor Vta1p/LIP5 (Shiflett et al.,2004; Lottridge et al., 2006; Azmi et al., 2008; Davieset al., 2010) ontomembrane-boundESCRT-III complexes.Vps4p/SKD1 is the only ATP-consuming enzymewithinthe core ESCRT machinery. Consistent with its centralrole in the ESCRT pathway, its activity is controlled bymultiple mechanisms. Vta1p/LIP5 increases in vitroVps4p/SKD1 ATPase activity in yeast, animals, andplants (Lottridge et al., 2006; Haas et al., 2007; Vild et al.,2015). In yeast, the C-terminal domain of Vta1p binds toVps4p, enhancing its oligomerization (Azmi et al., 2006;Lottridge et al., 2006; Yang and Hurley, 2010; Norganet al., 2013; Davies et al., 2014). Besides the direct acti-vationof SKD1/Vps4pbyLIP5/Vta1p, the coreESCRT-IIIproteins help recruit and activate theVps4p/SKD1ATPasecomplex (Saksena et al., 2009; Davies et al., 2010) by sub-strate engagement. Finally, the ESCRT-III accessory subu-nits Did2p/CHMP1, Vps60p/CHMP5, and IST1 also aidin the recruitment of Vps4p/SKD1 to the endosomalmembrane and interact directly with Vta1p/LIP5 (Azmiet al., 2008; Dimaano et al., 2008; Agromayor et al., 2009;Bajorek et al., 2009; Vild et al., 2015).

Whereas a role of Vta1p/LIP5 as a stimulator ofVps4p/SKD1 ATPase activity has been widely dem-onstrated, the mechanisms of action of the ESCRT-IIIaccessory subunits in MVB sorting have been harder todecipher. For example, IST1 is a divergent ESCRT-IIIsubunit that interacts with Did2p/CHMP1 and Vps4p/SKD1 in yeast and mammals but is not required forproper MVB sorting (Dimaano et al., 2008; Rue et al.,2008; Agromayor et al., 2009; Tan et al., 2015). However,the simultaneous deletion of IST1 and Vta1p/LIP5 en-hanced trafficking defects in both yeast and mammalian

cells (Dimaano et al., 2008; Rue et al., 2008; Agromayoret al., 2009), indicating a positive regulatory role of IST1in MVB sorting. In yeast, however, the effect of Ist1p onVps4p changes according to Ist1p concentration. At lowconcentration, Ist1p positively regulate recruitment ofVps4p through its interaction with Did2p but at highconcentration, Ist1p forms a heterodimer with Vps4p,blocking the interaction between Vps4p and the rest ofthe ESCRT machinery (Dimaano et al., 2008; Jones et al.,2012). In addition, supporting a negative role in theMVBpathway, Ist1p reduces the in vitro ATPase activity ofVps4p (Dimaano et al., 2008).

Although the ESCRT system is conserved across eu-karyotes, plants show an important diversification ofESCRT subunits, with the evolution of multiple iso-forms for most ESCRT components as well as plant-specific ESCRT proteins, such as FYVE DOMAINPROTEINREQUIRED FOR ENDOSOMAL SORTING1and POSITIVE REGULATOR OF SKD1 (PROS; Gaoet al., 2014, 2015; Reyes et al., 2014). The function ofSKD1 and some of its key regulators have been ana-lyzed in Arabidopsis. The over-expression of an ATP-locked form of Arabidopsis SKD1 results in alterationsof the endomembrane system (Cai et al., 2014) and leadsto a lethal phenotype in plants (Haas et al., 2007)whereas its specific expression in trichomes causesvacuole fragmentation and the presence of multiplenuclei (Shahriari et al., 2010). Mutations in the twoArabidopsis CHMP1 gene copies lead to embryo andseedling lethality and drastic mislocalization of theauxin efflux facilitators PIN1 and PIN2 (Spitzer et al.,2009). PROS, a plant-specific positive regulator of SKD1ATPase activity, is required for normal growth and cellexpansion (Reyes et al., 2014). Arabidopsis LIP5, as itsanimal and yeast counterparts, positively regulates SKD1enzymatic activity in vitro (Haas et al., 2007). In contrastto chmp1a chmp1b and PROS knock-down lines, lip5mutants show relatively minor growth defects. How-ever, LIP5 is a critical element in plant basal defenseagainst pathogens (Wang et al., 2014).

How the coordinated action of SKD1 regulators affectmembrane trafficking and development in multicellularorganisms is unknown. Here we investigate the functionof two SKD1 regulators, LIP5 and IST1, in Arabidopsis.We have found that LIP5 is important for controlling theconstitutive degradation of plasma membrane proteinsand the formation of ILVs. We also found a drastic evo-lutionary diversification of IST1-LIKE (ISTL) proteins inplants and investigated their interactions with LIP5 andother ESCRT components.

RESULTS

PIN Auxin Efflux Facilitators Are Mislocalized inlip5 Mutants

To investigate the function of LIP5 in endosomaltrafficking, we used two Arabidopsis lines with T-DNAinsertion in the LIP5 gene, lip5-1 (SAIL_854_F08) andlip5-2 (GABI_351F05; Supplemental Fig. S1A). Both

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lines were previously characterized as transcript-nullmutants (Haas et al., 2007; Wang et al., 2014) withslightly smaller rosette leaves when grown in soil (Wanget al., 2014) but otherwise able to complete their lifecyclenormally.Previous work (Spitzer et al., 2009) showed that

ESCRT proteins are important for the polar distributionand vacuolar degradation of PIN proteins. Therefore, weexpressed PIN2-GFP and PIN3-GFP in lip5 mutants un-der their native promoters and analyzed their localiza-tion and abundance by confocal microscopy imaging.We first analyzed the plasma membrane distribution ofPIN2-GFP in vertically grown lip5-1 roots and PIN3-GFPin lip5-1 columella cells before and after gravistimulation,since PIN3 polarization is enhanced toward the lowerside of the columella cells as part of the root gravitropicresponse (Friml et al., 2002; Abas et al., 2006; Harrisonand Masson, 2008; Kleine-Vehn et al., 2010). We foundthat lip5-1 root cells are able to polarize both PIN2-GFPand PIN3-GFP (Supplemental Fig. S2).However, although the polar localization of PIN2-

GFP did not seem to be affected, lip5-1 mutant roots

over-accumulated PIN2-GFP in both the plasmamembrane and tonoplast (Fig. 1, A and B). We werealso able to confirm the over-accumulation of PIN2-GFP in the lip5-1 roots by western blotting of proteinextracts (Fig. 1C). To test whether the accumulation ofPIN2-GFP was due to higher expression of the Pro-PIN2::PIN2-GFP transgene in the lip5-1 background, weperformed quantitative RT-PCR and found that thesteady-state levels of PIN2-GFP transcripts were similarin both wild-type and lip5-1 seedlings (Fig. 1D), indi-cating that the accumulation of PIN2-GFP in lip5-1 wasdue to a post-transcriptional event.

The lip5-1 mutation also caused the mis-sorting ofPIN3-GFP to the tonoplast (Fig. 1E). An initial analysisof PIN3-GFP fluorescence indicated that PIN3-GFP inlip5-1 was present in more cell tiers of the root capcolumella compared to wild type (Fig. 1, F and G).However, the extra tiers of cells presenting GFP signalin lip5-1 had most of PIN3-GFP at the tonoplast and notin the plasma membrane (Fig. 1F; asterisks), suggestingthat these peripheral columella cells do not expressPIN3-GFP any longer but fail to target the already

Figure 1. lip5-1 seedlings mis-sort and accumulate PIN proteins. A, Epidermal root cells in wild-type and lip5-1 seedlingsshowing the localization of PIN2-GFP. Arrows indicate PIN2-GFP mis-sorted to the tonoplast in lip5-1. WT: wild type. B,Quantification of PIN2-GFP signal intensity in epidermal root cells (n = 11 wild-type roots and 10 lip5-1 roots). Asterisks indicatesignificant differences between wild type and lip5-1 based on a two-tailed Student’s t-test (* P , 0.05, ** P , 0.001). Error barsindicate SD. C, Western-blot analysis of PIN2-GFPabundance in extracts from wild-type and lip5-1 roots. HISTONE3 was used asloading control. H3: HISTONE3;WT: wild type. D, Quantitative PCR of PIN2-GFP transcripts. Expression was normalized to thatof UBC9. Data are from three biological replicates. Error bars indicate SD. E, Root caps from wild-type and lip5-1 seedlingsshowing localization of PIN3-GFP in columella cells. Arrows indicate mis-sorting of PIN3-GFP to the tonoplast in lip5-1 colu-mella cells. WT: wild type. F, PIN3-GFP (green) in root cap cells of wild-type and lip5-1 seedlings. Propidium iodide (red) wasused for counterstaining. Arrowheads indicate tiers of cells with PIN3-GFP signal found at the plasma membrane; asterisks in-dicate tiers of cells within the root cap with PIN3-GFP signal detectable at tonoplast but not at the plasma membrane. WT: wildtype. G, Quantification of tiers of cells within the root cap with detectable PIN3-GFP signal, either at the plasma membrane,tonoplast, or both. (n = 9 wild-type and 10 lip5-1 roots). The asterisk (*) indicates significant differences between lip5-1 and wildtype based on a two-tailed Student’s t-test (P , 0.005). Error bars indicate SD. Bars = 20 mm.

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existing PIN3-GFP pool to the vacuolar lumen fordegradation.

LIP5 Affects Root Gravitropism But Not Root Growth

Root gravitropism depends on dynamic changes inthe amounts and localizations of plasma membrane-localized auxin efflux facilitators of the PIN family(Spalding, 2013). As we detected abnormal distributionof PIN2 and PIN3 in lip5-1, we asked whether lip5mutant roots were able to respond normally to gravity.Using a machine vision platform for measuring rootgrowth rate and direction (Miller et al., 2007; Brookset al., 2010), we found that the tips of both lip5-1 andlip5-2 roots reoriented faster than wild type aftergravistimulation (Fig. 2A). The faster root tip reorientationwas not due to a faster root elongation rate (SupplementalFig. S3).

LIP5 Affects Constitutive But Not Gravistimulation-Induced Degradation of PIN2

We then analyzed whether abnormal dynamics ofPIN proteins during gravistimulation were responsi-ble for the enhanced root gravitropic response in lip5seedlings. During gravistimulation, PIN2 is preferen-tially degraded in the epidermis of the upper side of theroot zone, presumably helping to establish the appro-priate auxin differential between the upper and lowersides of the elongation zone that produces curvature

(Abas et al., 2006). Degradation of PIN2 during gravi-tropism depends on PIN2 ubiquitination, endocyticinternalization, and vacuolar degradation (Leitner et al.,2012a, 2012b). To assess the ability of lip5 mutantsto degrade PIN2, we imaged lip5-1 and wild-typeseedlings expressing PIN2-GFP 150 min after grav-istimulation and quantified the fluorescence intensity atthe plasma membrane of the upper and lower sides ofthe root epidermis. We did not detect significant dif-ferences between lip5-1 and wild-type seedlings in theirability to remove PIN2-GFP from the plasma mem-brane at the upper side of the root (Fig. 2B). To furtherconfirm that the lip5 mutation does not affect endocy-tosis rates from the plasma membrane, we monitoredthe internalization of plasma membrane-bound FM4–64 in lip5 and wild-type seedlings and found no differ-ences in FM4 to FM64 endocytosis rates (SupplementalFig. S4).

If endocytosis rates are normal in lip5 plants, wereasoned that later steps in the endosomal pathwayand degradation of PIN proteins could be affected.We measured PIN2-GFP abundance during grav-istimulation of wild-type and lip5-1 mutant seedlings.Consistent with previous studies (Kleine-Vehn et al.,2008), PIN2-GFP abundance in wild-type seedlingsincreased by more than 2-fold at 60 min (T1) aftergravistimulation, followed by a sharp reduction of ap-proximately 50% at 150 min (T2). At 210 min aftergravistimulation (T3), the abundance of PIN2 was restoredto T1 levels (Fig. 2, C and D). Although the lip5-1 rootscontained twice as much PIN2-GFP compared to wild

Figure 2. Root gravitropism and degradation ofPIN2-GFP in lip5 mutants. A, Tip-angle timecourse of seedlings of wild-type and lip5 allelesafter 90˚ reorientation (gravistimulation). Thegraph shows the average tip angle at 2-min inter-vals mean 6 SE for each genotype. Significant dif-ferences (P, 0.05) in tip angle between wild-typeand lip5 mutants are shown in dashed lines asupward deflections (n = 16 seedlings from eachgenotype). B, PIN2-GFP abundance at the plasmamembrane and intracellular membranes includingtonoplast of epidermal cells 150 min after grav-istimulation. The graph shows ratios PIN2-GFPsignal intensity between the lower and upper sideof the gravistimulated roots (n = 18–21 seedlingsfor each genotype). Bars indicate SD. IC: intracel-lular; PM: plasma membrane; WT: wild type. C,Western-blot analysis of PIN2-GFP abundance inextracts from gravistimulated wild-type and lip5-1roots. HISTONE3 was used as loading control.H3: HISTONE3; WT: wild type. D, Densitometricquantification of PIN2-GFP/H3 ratios based onthree independent western blots. WT: wild type. E,Immunolocalization of PIN2-GFP on wild-typeand lip5-1 MVBs. Arrowheads indicate the pres-ence of 6-nm gold particles as indication of posi-tive labeling. WT: wild type. F, Red dots wereoverlaid on the images displayed in E to moreeasily visualize the 6-nm gold particles. WT: wildtype. Bars = 200 nm.


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type before gravistimulation, the relative changes inprotein abundance after gravistimulation, including adecrease of approximately 50% at 150 min, were verysimilar to those observed in wild-type roots (Fig. 2, Cand D). Consistently, we did not detect a significantincrease in the PIN2-GFP pool mis-sorted to thetonoplast in the upper-side root epidermal cells by thetime PIN2-GFP was being degraded in their vacuoles(150 min after gravistimulation; Fig. 2B). These resultssuggest that lip5-1 roots mis-sorted PIN2-GFP to thetonoplast during its constitutive endosomal-mediateddegradation.However,we could not detect alterations ingravistimulation-triggeredPIN2proteolysis in lip5-1 roots.To determine whether cargo is sorted properly into

ILVs of lip5-1 MVBs, we immune-localized PIN2-GFPon root cells. We detected positive labeling on ILVs ofboth wild-type and lip5-1 MVBs (Fig. 2, E and F), indi-cating that lip5-1 ILVs contain cargo from the plasmamembrane.

IST1-LIKE Proteins in Arabidopsis

Whereas LIP5 and other ESCRT components posi-tively modulate SKD1 ATPase activity, IST1 is the onlyprotein proposed to act as an SKD1 negative regulator.To understand the functional integration of positive

and negative regulators for the key ESCRT componentSKD1, we decided to investigate the function of IST1in plants. Through a BLAST search, we identified theArabidopsis proteinwith highest homology to the yeastIST1 domain (33% identity, AT1G34220) and called itISTL1. We expressed recombinant ISTL1–63HIS andGST-LIP5 in bacteria and found they are able to interactin vitro (Fig. 3A). However, a more careful analysis ofour IST1 domain BLAST search indicated that, contraryto most eukaryotic organisms containing only one IST1gene copy, Arabidopsis contains 12 genes coding forIST1-domain-containing or ISTL proteins (Fig. 3B). Wealso identified three IST1-like genes in the moss Phys-comitrella patens and five IST1-like genes in rice (Oryzasativa), suggesting an early diversification of ISTL genesin the plant lineage. In a phylogenetic analysis of ISTLprotein sequences, we found that only ArabidopsisISTL1 to ISTL6 have rice counterparts, whereas ISTL7to ISTL12 grouped into their own clade (Fig. 3C).

We decided to focus on the ISTL1–6 group and testedwhether they are all part of the ESCRT machinery byassessing their capability to interact with LIP5, SKD1,and CHMP1 in directed yeast-2-hybrid (Y2H) assays(Fig. 4). As the expression of full-length LIP5 resulted inautoactivation of the GAL4 expression system, we useda fragment of LIP5 (amino acids 1–159) containing two

Figure 3. ISTL proteins in Arabidopsis. A, In vitro pull-down assay of recombinant Arabidopsis LIP5 and ISTL1 proteins taggedeither with GST or 63His and expressed in bacteria. GST-LIP5 or GST alone bound to beads were incubated for 2 h with His-ISTL1. Beads were washed three times. Input and output fractions were loaded on an SDS-PAGE gel and transferred to a nitro-cellulose membrane. Proteins were detected with specific antibodies anti-GSTor anti-63His. I: input; O: output. B, Diagrams ofIST1 proteins in humans and yeast and ISTL1 proteins in Arabidopsis. Highlighted in yellow is the position of the characteristicIST1 domain. C, Phylogenetic analysis of IST1-LIKE performed with MEGA6. Bootstrap values are shown above each branch.Scale indicates 0.5 amino-acid substitutions per site. Pp, Physcomitrella patens; At, Arabidopsis thaliana; Os, Oryza sativa; Sc,Saccharomyces cerevisiae; Hs, Homo sapiens; Tb, Trypanosoma brucei; Ls, Leishmania major.





microtubule interacting and trafficking domains ex-pected to interact with IST1 proteins (Agromayor et al.,2009; Guo and Xu, 2015). As evidence of positive in-teraction, we determined general growth of yeast cellson interaction-selective plates (Fig. 4A), b-galactosidaseactivity on a colony filter lift assay (Fig. 4B), and theratio between colonies grown on interaction selectionmedia and transformation selection media for interac-tions with positive b-galactosidase activity (Fig. 4C; see“Materials andMethods”). In the three tests, only ISTL1and ISTL5 showed consistent strong interactions withLIP5(1–159), CHMP1, and SKD1whereas ISTL4 showedpositive interaction with CHMP1 but not with SKD1 orLIP5 (1–159; Fig. 4).

Within the ISTL1–6 group, ISTL4 and ISTL5 areunique in being approximately twice as large as theother four proteins and preferentially expressed inpollen (Fig. 3B; Supplemental Fig. S5). Therefore, wenarrowed our functional analysis to ISTL1, 2, 3, and 6,which are broadly expressed in both vegetative andreproductive tissues (Supplemental Fig. S5). We iden-tified T-DNA lines with insertions in all four genes andisolated the istl1-1, istl2-1, istl3-1, and istl6-1 mutant

alleles (Fig. 5A). All four single istl mutants grew nor-mally in soil with no obvious developmental defects(Fig. 5B). Since the four genes have overlapping ex-pression patterns (Supplemental Fig. S5), we suspectedthey could act redundantly. Therefore, we combined allfour transcript-null mutations in an istl1 istl2 istl3 istl6quadruple mutant (Fig. 5C). However, the quadruplemutant plants also showed normal growth and devel-opment (Fig. 5C).

A Genetic Interaction between LIP5 and ISTL1 ControlsDefense Responses and Fertility in Arabidopsis

The deletion of either IST1 or LIP5/Vta1 causes mildendosomal trafficking defects in yeast and animal cells.However, the deletion of both genes simultaneouslyresults in severe mis-sorting of MVB cargo proteins(Dimaano et al., 2008; Rue et al., 2008; Agromayor et al.,2009). To test whether this synthetic genetic interactionis conserved in plants and to uncover functional re-dundancy among ISTL genes, we crossed every one ofthe istl mutants with lip5-1. Double mutants for lip5-1and istl2, istl3, or istl6 were indistinguishable from

Figure 4. Y2H assays between ISTL1–6 proteins and LIP5(1–159), CHMP1, and SKD1. A, Y2H assays between ISTL1–6 and LIP5(1–159), CHMP1, or SKD1. Plates show colonies growing on 2LWH medium (selection for interaction) and on 2LW medium(selection for transformation). Controls were performed by coexpressing ISTL, LIP5(1–159), CHMP1, and SKD1 with the corre-sponding prey or bait empty vectors. B, Detection of b-galactosidase (GUS) activity by a colony filter lift assay using X-gal as asubstrate. Asterisks (*) indicate positive GUS activity detection. C, Quantification of colonies grown on selection and interactionmedia for those cases with positiveGUS activity. Graphs show ratios between the number of colonies grown on2LWHmediumand the number of colonies grown on2LWmedium. The ratio values from control plateswere subtracted from those testing directinteractions between ISLT proteins and LIP5(1–159), CHMP1, or SKD1. Between 250 and 400 colonies were counted in eachcase. The graphs show combined results from two independent experiments; error bars indicate SD.


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single lip5-1 mutant plants (Fig. 5D). However, thecombination of lip5-1with istl1-1 resulted in plants withreduced growth, small rosette leaves, and shorter pet-ioles. These plants showed signs of early senescenceafter approximately 3 weeks in soil at 22°C (Fig. 5, Dand E). We also found that the istl1-1 lip5-1 cotyledonssenesced earlier than those of single mutants or wild-type plants grown in the same conditions (Fig. 6A). Todetermine the presence of dead cell sectors and accu-mulation of H2O2 in leaves, we stained leaf sampleswith trypan blue and DAB (3,39-diaminobenzidine;Shirasu et al., 1999; Noutoshi et al., 2005; Ichimura et al.,2006), respectively (Fig. 6, B and C).We found that istl1-1lip5-1 leaves stained more strongly with both methodsthan singlemutants andwild-type leaves (Fig. 6, B andC).Dwarfism, late lethality, early senescence of both

cotyledons and leaves, and spontaneous cell death arecommon features of mutants exhibiting constitutivedefense response (Lam, 2004; Ichimura et al., 2006), thatis, the up-regulation of genes involved in pathogen re-sponses in the absence of pathogens or elicitors. In thesecases, the constitutive up-regulation of defense-relatedgenes leads to spontaneous cell death and oxidativestress, even in the absence of pathogens. To testwhether the phenotypic defects of the istl1-1 lip5-1wereconnected to a constitutive pathogen response, we an-alyzed the accumulation of PATHOGEN-RELATED1(PR1), a salicylic acid and defense response markergene (Bowling et al., 1994; van Loon et al., 2006; Tsudaet al., 2008). We found a strong up-regulation in theexpression of PR1 in the double ist1-1 lip5-1 comparedto that in wild-type and lip5-1 seedlings (Fig. 6D).High temperatures have been shown to partially

or completely suppress dwarfism in mutants exhibitingconstitutive defense responses (Bieri et al., 2004;Ichimura et al., 2006; Zhu et al., 2010; Wang et al., 2011).We found that the dwarfism of istl1-1 lip5-1 plants was

partially suppressed at 28°C, and that mutant plantsoutlived by 4 weeks their counterparts grown at 22°C(Fig. 6E). Consistently, istl1-1 lip5-1 plants grown at 28°Cshowed highly reduced levels of PR1 transcripts com-pared to plants grown at 22°C (Fig. 6D). Although istl1-1lip5-1 mutant plants grown at 28°C were able to forminflorescences, they were unable to produce seeds.

ISTL1 and LIP5 Control ILV Size and MVB Formation

To determine whether the lack of both LIP5 andISTL1 function affects MVB formation, we ana-lyzedMVBmorphology in high-pressure frozen/freeze-substituted wild-type, lip5-1, istl1-1, and lip5-1 istl1-1MVBs (Fig. 7A). When the identification of MVBswas not possible solely on morphological recognition,we used immunogold labeling with antibodies againstMVB-localized proteins such as RABF2A or VSR1(Haas et al., 2007; Gao et al., 2014). We found that thediameter of MVBs in the four genotypes was similar,with mean diameters ranging from 260 nm to 280 nm(Fig. 7B), but lip5-1 and istl1-1 lip5-1 MVBs containfewer intraluminal vesicles compared to those of wildtype and istl1-1 (wild type, 5 ILV/MVB section, SD 62.4, n = 49 MVBs; lip5-1, 2 ILV/MVB section, SD 6 1.2,n=87MVBs; istl1-1, 4.3 ILV/MVBsection, SD6 2.8,n=44;istl1-1 lip5-1, 1.4 ILV/MVB section, SD6 1.3, n= 41MVBs).This represents approximately 60% and 70% decreasein the number of ILVs in lip5-1 and istl1-1 lip5-1 MVBs,respectively. Interestingly, whereas the ILVs fromwild-type, istl1-1, and lip5-1 MVBs are similar in sizewith mean diameters ranging between 35 nm and38 nm (Fig. 7B), the double istl1-1 lip5-1 mutant ILVswere approximately 27% larger in diameter (mean di-ameter = 45 nm, SD 6 10.7 nm, n = 40) and conse-quently, 52% larger in surface area compared to theother three genotypes (Fig. 7B). These results indicate

Figure 5. Identification and characteriza-tion of istl mutants. A, Diagrams of ISTLgenes and position of T-DNA insertion inthe analyzed mutants. Black rectanglesindicate exons; black lines indicate introns.B, Overview of 26-d-old wild-type andsingle istl mutant plants grown in soil. C,Overview of 26-d-old wild-type and qua-druple istl1 istl2 istl3 istl6 (quad) mutantplants; RT-PCR of ISTL1, 2, 3, and 6 tran-scripts from wild-type and quad mutantplants. D, Overview of 26-d-old singlelip5-1 and double mutant combinationsbetween lip5-1 and istlmutations. E, Close-up view of 26-d-old istl1 lip5-1 doublemutants.





that LIP5 is necessary for ILV formation, and that to-gether LIP5 and ISTL1 control ILV size.

DISCUSSION

We have found that the ESCRT component LIP5is critical for the constitutive degradative sorting ofplasma membrane-localized auxin efflux facilitators ofthe PIN family, but not for the gravity-induced degra-dation of PIN2. We also found that the enhancedgravitropic response observed in lip5 roots is likely dueto changes in the abundance of PIN2 at the plasmamembrane. We investigated six of the 12 ArabidopsisISTL proteins and showed that, among the ISTL1–6group, ISTL1 showed robust interactions with LIP5,CHMP1, and SKD1 in a Y2H system.Consistently, ISTL1shows a strong synthetic interaction with LIP5 that neg-atively regulates spontaneous cell death, early lethality,and constitutive defense responses.

ESCRT, Gravitropism, and PINs

Root gravitropic response provides exceptional toolsto analyze endosomal protein trafficking since it in-volves drastic changes in the distribution and deg-radation rates of plasma membrane-localized PINproteins. Auxin redistribution to the lower side of theroot upon gravistimulation depends on changes in thedistribution and abundance of PIN efflux facilitators(Friml et al., 2002; Harrison and Masson, 2008; Kleine-Vehn et al., 2010). PIN proteins undergo constitutiveendosomal recycling (Geldner et al., 2003; Abas et al.,2006; Dhonukshe et al., 2007; Kleine-Vehn et al., 2008,2011) and are targeted to ILVs and vacuolar degrada-tion by the ESCRT machinery (Spitzer et al., 2009).

Either enhanced PIN3-GFP polarization toward thelower side of the columella cells or an expansion of its

expression domain within the root cap could relate toenhanced root gravitropism. We did not observe anincrease in the number of root cap cells with PIN3-GFPat the plasma membrane (Fig. 1F), but we noticed aslight increase in the number of vertically grown rootsshowing lateral polarization of PIN3-GFP in lip5 seed-lings (Supplemental Fig. S2). However, how this in-crease in the random lateral polarization of PIN3-GFPin non-gravistimulated roots can affect gravitropism, isunclear.

Consistent with PIN2 being an ESCRT cargo (Spitzeret al., 2009), PIN2 ubiquitination is important for both itsconstitutive and gravistimulation-related vacuolar deg-radation (Leitner et al., 2012a, 2012b). The localizationand endosomal recycling rates of PIN2 depend on auxinand other hormones (Paciorek et al., 2005; Willige et al.,2011; Löfke et al., 2013;Marhavý et al., 2014), light (Laxmiet al., 2008; Wan et al., 2012), temperature (Shibasakiet al., 2009; Hanzawa et al., 2013), cell types (Löfke et al.,2015), and gravity (Abas et al., 2006; Rahman et al., 2010).

We have found that lip5 mutant seedlings respondfaster to reorientation of root growth induced bygravistimulation. In lip5-1 root cells, PIN2-GFP was notonly mis-sorted to the vacuolar membrane, but alsoover-accumulated at the plasma membrane. Althoughnot analyzed in this study, loss-of-function of LIP5could affect the abundance and distribution of manyother plasma membrane proteins that control auxindistribution in roots [e.g. the AUXIN RESISTANT1 in-flux carrier (Marchant et al., 2002), other PIN proteins(Blilou et al., 2005; Wisniewska et al., 2006), and mem-bers of the ATP BINDING CASSETTE SUBFAMILY Bfamily (Noh et al., 2001; Lewis et al., 2007; Titapiwata-nakun et al., 2009; Cho et al., 2014)]. However, the soleincrease of PIN2 abundance at the plasma membranehas been shown to enhance root gravitropic response.For example, enhanced gravity root responses have

Figure 6. Early cotyledon senescence and spon-taneous cell death in istl1 lip5 mutants. A, Coty-ledons from 3-week-old plants. WT: wild type. B,First rosette leaves from 3-week-old plants stainedwith DAB. WT: wild type. C, First rosette leavesfrom 3-week-old plants stained with Trypan Blue.WT: wild type. D, Quantitative PCR of PR1 tran-script. Expression was normalized to that of PP2A.Bars indicate mean 6 SE. WT: wild type. E, Partialsuppression of istl1 lip5-1 of growth defects at ahigh temperature. A quantity of 31-d-old plantsgrown at 22˚C or 28˚C for 17 d. WT: wild type.


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been associated with increased accumulation of PIN2at the plasma membrane due to (1) enhanced PIN2endosomal recycling associated with gibberellic acidaccumulation (Löfke et al., 2013) or high temperature(Hanzawa et al., 2013); (2) reduced PIN2 endocytosis inROP6 gain-of-function lines (Chen et al., 2012); and (3)stabilization of PIN2 at the plasma membrane by over-expression of the tonoplast transporter ZINC INDUCEDFACILITATOR-LIKE1.1 (Remy et al., 2013). Thus, al-though it is reasonable to think that enhanced gravi-tropism in lip5 roots could be due to the combinedchanges in abundance and localization of many plasmamembrane proteins, the over-accumulation of PIN2 atthe plasma membrane itself could explain the observedroot-tropic responses to gravity.Why would a mutant with normal endocytosis but

abnormal MVB function have more PIN2 at the plasmamembrane? It is logical to hypothesize that the strongreduction in ILV formation in lip5 mutants leads to re-duced sequestration of PIN2 into the lumen of MVBsand its accumulation at the MVB limiting membrane.As plasma membrane proteins are in continuousendocytic and recycling fluxes through the endomem-brane system, the failure to efficiently sequester PIN2into ILVs would likely affect PIN2 recycling rates aswell. The endosomal recycling of PIN2 depends on theretromer proteins SORTIN NEXIN1 and VACUOLARPROTEIN SORTING29 (Jaillais et al., 2006, 2007;Kleine-Vehn et al., 2008; Hanzawa et al., 2013). The rateof PIN2 recycling can be affected by environmentalfactors. High temperature has a positive effect on theSORTIN NEXIN1-dependent PIN2 recycling from lateendosomes and, similar to the lip5-1 mutation, leads toenhanced root gravitropic responses (Hanzawa et al.,2013). Thus, it is possible that the deficient sorting ofPIN2 into the lip5 MVB lumen leads to more PIN2

available for recycling and an accumulation of PIN2 atthe plasma membrane.

Whereas we found that lip5-1 roots grown verticallyover-accumulate PIN2-GFP in both the plasma mem-brane and tonoplast, we did not detect defects in thegravity-triggered degradation of PIN2-GFP. Both byconfocal imaging and western blotting, we measuredsimilar patterns of PIN2-GFP dynamics in wild-typeand lip5-1 roots after gravistimulation. The accumula-tion of PIN2-GFP in vertically grown roots is likely theresult of the cumulative endosomal mis-sorting ofPIN2-GFP during its constitutive degradation. It ispossible that gravity-induced PIN2 proteolysis alsocarries some degree of cargo mis-sorting at endosomesas the constitutive degradation pathway, but it is notstrong enough to be detected with our tools. Alterna-tively, other components regulating MVB sorting couldact in situations when rapid proteolysis of plasmamembrane proteins is required, overriding the need forLIP5. In either case, LIP5 seems to be more critical forthe constitutive turnover of PIN2 than for its gravity-induced proteolysis, suggesting that the two proteolyticprocesses may be regulated differently.

Diversification of ISTL Genes in Arabidopsis

Yeast IST1 consists of an N-terminal IST1 domain ofapproximately 150 amino acids that binds CHMP1 and aC-terminal region that binds the N-terminal domain ofVps4p/SKD1. In yeast, the single vta1D, vps60D, and ist1Dmutant strains exhibit either mild or undetectable MVBsorting defects. However, simultaneous deletion of IST1and either Vta1p or Vps60p leads to strongmis-sorting ofendosomal cargo to the vacuolar membrane. It has beenpostulated that IST1 and Did2p/CHMP1 form a complexthat positively regulates Vps4p/SKD1ATPase activity, in

Figure 7. Analysis of MVBs in roots. A,Overview of multivesicular bodies in high-pressure frozen/freeze-substituted roots.WT: wild type. B, Quantification of morpho-logical features ofMVBs (n=41–87MVBs and40–200 ILVs from at least two roots for eachgenotype). Letters above the bars representstatistical significance (one-way ANOVA fol-lowedbyTukey,P,0.05); bars sharing a letterare not significantly different fromoneanother.Bars indicate SD. Bars = 200 nm. WT: wildtype.





concert or redundantly with the Vta1p/LIP5–Vps60pcomplex (Dimaano et al., 2008; Rue et al., 2008).

We have identified 12 genes in Arabidopsis coding forpredicted proteins with conserved IST1 domains. Wefound that ISTL1 interacted strongly with both CHMP1and SKD1 in Y2H assays. Consistently, ISTL1, but notISTL2, 3, or 6, showed a strong genetic interaction withLIP5. These results suggest that, out of the analyzed ISTLgenes, only ISTL1plays a central role in theMVBpathway.The synthetic effects betweenmutations in LIP5 and ISTL1resulted in reduced growth, late lethality, and spontane-ous cell death due to a constitutive pathogen response asevidenced by the strong up-regulation of the PR1 gene.Interestingly, although previous studies have shown thatLIP5 is important for signaling in response to pathogens(Wang et al., 2014), lip5 mutants do not show up-regulation of PR1, early senescence, or spontaneous celldeath, suggesting that either LIP5 or ISTL1 is sufficient tonegatively regulate constitutive pathogen responses. Sev-eral plasma membrane-localized receptor-like kinaseswith important functions in plant development andgrowth have been implicated in constitutive pathogenresponses. Thus, BRASSINOSTEROID INSENSITIVE1-ASSOCIATED RECEPTOR KINASE1, SOMATICEMBRYOGENESIS RECEPTOR KINASE4, and BRASSI-NOSTEROID INSENSITIVE1-ASSOCIATEDRECEPTORKINASE1-INTERACTING RECEPTOR-LIKE KINASE1negatively regulate spontaneous cell death and seedlinglethality, whereas CYS-RICH RECEPTOR-LIKE KINASEacts as positive regulators (de Oliveira et al., 2016). It willbe interesting to investigate in future studies whether ab-normal trafficking of some of these proteins in the doublemutant can explain the observed phenotypes.

Plants have evolved new proteins with ESCRT func-tion such as FYVEDOMAINPROTEINREQUIREDFORENDOSOMAL SORTING1 (Gao et al., 2014, 2015) andPROS (Reyes et al., 2014) and increased the number ofESCRT-like subunits, some of which do not seem to actin the MVB pathway. As an example, three genes codefor VPS2-like proteins in Arabidopsis, VPS2.1, VPS2.2,and VPS2.3 (Winter and Hauser, 2006). Different fromVPS2.1, which seems to play a bona fide VPS2 function,the other two Arabidopsis VPS2 proteins are neither ableto interact with the ESCRT-related AMSH3 deubiquiti-nase enzyme nor to be recruited to endosomes when anATP-locked form of SKD1 is expressed in protoplasts(Katsiarimpa et al., 2011). A similar scenario may befound in the Arabidopsis ISTL gene family. WhereasISTL1 seems to act as a canonical IST1 ESCRT-III subunit,the other ISTL proteinsmay have tissue-specific roles, forexample ISTL4 and ISTL5 that are primarily expressed inpollen, or that may have evolved ESCRT-independentfunctions.

Formation of MVB in Plants

Different from animal and plant cells, yeast cellslacking core ESCRT components have flattened stackedendosomes called “class E” compartments (Riederet al., 1996), making direct structural comparisons

between mutant MVBs in plants and yeast difficult. Incontrast, deletions of the yeast ESCRT-III-related sub-units cause relatively milder defects in MVB structure,mostly affecting ILV size and luminal membrane sur-face area (Nickerson et al., 2010). ILVs in yeast MVBsare smaller (mean diameter 24–27 nm; Nickerson et al.,2010; Adell et al., 2014) than those in plants (meandiameter 35–38 nm). However, the variation in ILVsizes due to mutations in LIP5/Vta1p, IST1, and otherESCRT-III accessory subunits shared some similarities.For example, both in plants and yeast, the single lip5/vta1D and istl1/ist1D mutations caused no changes inILV diameter. The double istl1 lip5mutant in plants andthe ist1D did2D doublemutant in yeast show an increasein approximately 30% of the ILV diameter, which interms of ILV membrane surface area translates into anincrease of 52% in plants and 68% in yeast.

However, the changes in size and number of ILVs donot seem to correlate directly with the severity of themis-sorting defects seen in ESCRT mutants. For exam-ple, the MVBs of Arabidopsis lip5 and chmp1a chmp1bmutants (Spitzer et al., 2009) are structurally similar,both with reduced number but normal-sized ILVs. Ifthe underlying cause of MVB sorting defects in bothmutants is the reduction in ILV surface area and con-sequently, the reduced capacity to accommodate MVBcargo in ILVs for degradation, both mutants shouldshow similar mis-sorting defects. However, whereasthe chmp1a chmp1b mutant shows strong mis-sorting ofplasma membrane to the tonoplast, and complete lossof PIN2 polarization, embryo, or early seedling lethalityas well as general abnormal development (Spitzer et al.,2009), lip5 plants showed partial mis-sorting of PIN2and PIN3 to the tonoplast, reduced growth of aerialparts, and normal lifecycle. We have detected abundantPIN2-GFP cargo on ILVs of both wild-type and lip5MVBs, whereas in PIN1-GFP it was detected in ILVs ofwild type but not of chmp1a chmp1b MVBs (Spitzer et al.,2009). This means that although both CHMP1 and LIP5are required for the formation of ILVs, CHMP1 but notLIP5 has a critical role in cargo sequestration into ILVs.

MATERIALS AND METHODS

Plant Material

The following lines were used in this study: lip5-1 (SAIL_854_F08) and lip5-2(GABI_315F05, Haas et al., 2007; Wang et al., 2014); istl1-1 (SALK_021562),istl2-1 (SALK_060592), istl3-1 (SAIL_84_C12), istl6-1 (SALK_101433), andProPIN2:PIN2-GFP (Laxmi et al., 2008); and ProPIN3:PIN3-GFP (Zádníkováet al., 2010). Fluorescent protein expression cassettes were introgressed into lossof function lines by crossing.

Unless otherwise stated, plants were grown in growth chambers at 22°C under16 h of light and 8 h of dark cycle. Seedswere pretreated in 70% ethanol for 10min,surface-sterilized in 50% bleach for 1min, andwashed in distilledwater for at leastfour times. Seedswere sowed onplates containing 0.53Murashige and Skoog saltssupplemented with 1% Suc, stratified at 4°C for 2–4 d, and set to germinate.

Quantitative PCR and RT-PCR

RNA was isolated from roots of 5-d-old wild-type and lip5-1 seedlingsexpressing PIN2-GFP or from rosette leaves of soil-grown wild-type, lip5-1, andistl1 lip5-1 plants using a Trizol reagent following the manufacturer’s instructions.


Buono et al.



Reverse transcription was carried out using the RETROscript kit (Ambion, FosterCity, CA). Amplification and detection were performed on a model no. MX3000PqPCR system (Stratagene, San Diego, CA) to monitor double-strand DNA syn-thesis. Each reaction contained 1 mL of cDNA, 0.5 mL of each of the two gene-specific primers, and 10 mL of MAXIMA SYBR Green/ROX qPCR Master Mix(Thermo Fisher Scientific, Foster City, CA) in a final volume of 20 mL. Results wereanalyzed with LinRegPCR (V. 2013.0; http://www.hartfaalcentrum.nl/index.php?main=files&sub=LinRegPCR). The relative value for expression levels of eachgene was calculated by the equation Y = 22DDCt (Livak and Schmittgen, 2001). Thecalculated relative expression values are normalized to the wild-type expressionlevels (wild type = 1). Two different technical replicates and three independent setsof plants were used for analysis. Primers used for quantitative PCR analysis arelisted in Supplemental Table S1.

Root Gravitropism

We used a morphometric method developed by Miller et al. (2007). Platescontaining 5-d-old seedlings were mounted vertically and transverse to theoptical axes of CCD cameras. Rotating the plate until the root tip was parallel tothe camera’s horizon (90°) initiated the gravitropic response. Each time series ofimages representing the gravitropic response of a single root was processed byan algorithm that determined the midline of the root in each frame and com-puted the root tip angle.

Confocal Laser Scanning Imaging

For FM4-64 internalization experiments, 5-d-old seedlings grown on plateswere incubated in 4mM FM4-64 (Molecular Probes, Invitrogen, Carlsbad, CA) in0.53Murashige and Skoog salts supplemented with 1% Suc for 5 min, washedin the samemediumwithout the dye, then mounted and imaged at 10 min afterinitial incubation with the dye. Seedlings were observed on a model no. LSM510 with a Plan-Apochromat 633 objective [NA = 1.4 oil-immersion, differen-tial interference contrast (DIC); both by Carl Zeiss, Jena, Germany]. A 488-nmargon laser line was used for excitation, and emission was collected using a LP560 filter (Carl Zeiss). Images with no saturated pixels and captured undersimilar microscope settings were analyzed with the aid of FIJI (Schindelin et al.,2012). Internalization of FM4-64 is presented as the ratio of fluorescence in-tensity at the cytoplasm normalized by the intensity at the plasma membrane.

For PIN2-GFP and PIN3-GFP imaging, 4-5-d-old mutant and control seed-lings grown on plates or directly on coverslips were observed by using a modelno. LSM 510 with a C-Apochromat 403 objective (NA = 1.2 water-immersion;Carl Zeiss) or Plan-Apochromat 633 objective (NA = 1.4 oil-immersion, DIC;Carl Zeiss), and a model no. LSM 780 with a Plan-Apochromat 633 objective(NA = 1.4 oil-immersion, DIC; Carl Zeiss). A 488-nm argon laser line was usedfor excitation, and emission was collected between 500 and 530 nm or 490 and562 nm.

Protein Purification and Interaction Assays

Recombinant GST-LIP5 and 63HIS IST1 LIKE (ISTL1) proteins wereexpressed in Escherichia coli BL21 and purified as described previously inSpitzer et al. (2009) and Reyes et al. (2014). For the in vitro interaction assay,equivalent amounts of the purified proteins were incubated 2 h at 4°C in a50-mM Tris-HCl, 0.1% Triton-X-100, protease inhibitor cocktail “Complete”(Roche Diagnostics, Indianapolis, IN), 1 mM sodium orthovanadate, pH 7.6 (i.e.Input). The glutathione-agarose beads were then rinsed three times with thesame buffer described above (i.e. Output). Samples were denatured usingLaemmli buffer, separated by SDS-PAGE and transferred onto nitrocellulosemembrane. The proteins were detected using commercial anti-63His and anti-GST antibodies (Sigma-Aldrich, St. Louis, MO).

Immunoblot Analysis

Seedling roots were frozen in liquid nitrogen and homogenized in a buffercontaining: 250 mM sorbitol; 50 mM HEPES-BPT at pH 7.4 (Sigma-Aldrich);25 mM ascorbic acid; 1 mM DTT (Fluka/Sigma-Aldrich); 6 mM EGTA 1.2%(w/v) Polyvinyl porrolidone-40 (Sigma-Aldrich); and protease inhibitor cock-tail “Complete” (Roche Diagnostics). A 1:2 (w/v) ratio of tissue and homoge-nization media was used. The homogenization media was filtered throughmiracloth and centrifuged at 10,000g for 15min. The supernatant was recoveredand kept aside. The pellet was extracted with 1/10 of the original volume ofhomogenization buffer used in the first extraction and then centrifuged at 600g

for 3 min. The supernatant from this second extraction was added to the firstsupernatant. The combined supernatants were mixed, centrifuged at 600g for3 min, and the cleared homogenate was retained as supernatant. These clearedsupernatants were mixed with acetone (1 mL of acetone per 200 mL of clearedhomogenate), incubated for 10 min at 220°C, and centrifuged at 20,000g for10 min. The resulting supernatant was discarded and the pellet was suspendedin 50 mL of resuspension buffer containing 250 mM sorbitol, 25 mM HEPES-BPTpH 7.4, 1 mM DTT, and protease inhibitor cocktail. Protein concentration wasmeasured with a DC Protein Assay (Bio-Rad Laboratories, Hercules, CA)according to manufacturer’s instructions.

For SDS/PAGE, we used a 5% (w/v) acrylamide stacking gel and 12% re-solving gel in a MiniProtean 3-Electrophoresis System (Bio-Rad Laboratories).Resuspended pellets aliquots containing 10 mg of protein were incubated for15 min at room temperature before loading on the gel. After electrophoresis,gels were either stained with Coomassie Brilliant Blue R-250 (Sigma-Aldrich) ortransferred to nitrocellulose membranes (GE Healthcare Lifesciences, Pitts-burgh, PA) for immunoblotting. Membranes were stained with 0.15% (w/v)Ponceau Red (Sigma-Aldrich) to check for protein transfer and then blockedwith 5% (w/v) dried nonfat milk in TBS (Tris-buffered saline) buffer with 0.1%TWEEN 20. The antibodies used for western-blot analysis revealed, in eachcase, single bands at the expected molecular masses. The primary antibodiesused were mouse anti-GFP (1:1000; Roche USA) and rabbit anti-H3 (1:5000;Abcam, Cambridge, UK). Blots were incubated with the appropriate IgG-HRP-conjugated secondary antibody. Antigen–antibody complexes were developedusing either electrochemiluminescence western-blotting substrate or SuperSignal West Femto Maximum Sensitivity Substrate (Pierce, Rockland, IL). Blotswere exposed for different times; exposures in the linear range of signal wereselected for densitometric evaluation. Optical densities of the immunoreactivebands were measured using FIJI (Schindelin et al., 2012).

Electron Microscopy and Immunolabeling

Root tips were high-pressure-frozen/freeze-substituted for transmissionelectron microscopy analysis as described previously in Spitzer et al. (2009).Briefly, roots tips from 1-week-old seedlings were dissected and frozen in amodel no. HPM 010 high-pressure freezer (Baltec, Canonsburg, PA). Sampleswere freeze-substituted in 0.2% glutaraldehyde plus 0.2% uranyl acetate inacetone at290° for 4 d in an AFS Automated Freeze Substitution device (Leica,Wetzlar, Germany) and embedded in Lowicryl HM20 (Electron MicroscopySciences, Hatfield, PA). Sections were mounted on formvar-coated nickel gridsand blocked for 20 min with a 5% (w/v) solution of nonfat milk in TBS con-taining 0.1% TWEEN 20. The sections were incubated in the primary polyclonalantibodies against RABF2A/RHA1 (Haas et al., 2007), anti-VSR (Rose Bio-technology, Winchendon, MA), or GFP (Torrey Pines Antibodies, San Diego,CA) for 1 h, rinsed in TBS containing 0.5% TWEEN20, and transferred to thesecondary antibody (anti-rabbit IgG 1:10) conjugated to either 15- or 6-nm goldparticles for 1 h. Controls lacked the primary antibodies.

Morphometric analysis of multivesicular bodies and intraluminal vesicleswas performed with FIJI (Schindelin et al., 2012).

Histochemistry

Two-week Arabidopsis plants were vacuum-infiltrated with 2.5 mM DAB(3,39-diaminobenzidine) solution, cleared in a solution of 80% (v/v) ethanol,20% (v/v) chloroform, and 0.15% (w/v) trichloroacetic acid for 24 h, andmounted in 50% (v/v) glycerol solution (Pogány et al., 2004). For detectionof cell death, we prepared a Trypan Blue stock solution consisting of 10 g ofphenol, 10mL of glycerol, 10mL of lactic acid, 10mL of distilledwater, and 0.02 g ofTrypan Blue (Sigma-Aldrich). The stock solution was diluted 1:2 with ethanol 96%(Pogány et al., 2009). Arabidopsis leaveswere incubated in the lactophenol-TrypanBlue working solution, boiled in a water bath for 1 min, incubated in the workingsolution for 24 h, and cleared with 1:2 lactophenol/ethanol.

Y2H Assays

CHMP1, LIP5(1–159), and SKD1 cDNAs were cloned in-frame with theGAL4 DNA binding domain in the vector pBD-GAL4 and ISTL1–6 into theGAL4 DNA activation domain of the vector pAD-GAL4 (Stratagene), respec-tively. Yeast cells were transformed using a lithium acetate-based protocoland grown on synthetic dextrose media. For two-hybrid assays, cells werecotransformed with two plasmids. The ability to drive the HIS3 reporter genewas assessed by growing transformants on selective medium lacking Trp, Leu,




http://www.hartfaalcentrum.nl/index.php?main=files&sub=LinRegPCR

http://www.hartfaalcentrum.nl/index.php?main=files&sub=LinRegPCR



and His (2LWH). To quantify the interaction between partners, the number ofcolonies grown on 2LWH medium (selection for interaction) was divided bythe number of colonies grown on 2LW medium (selection for transformation)to calculate the percentage of colonies showing interaction. The ratio valuesfrom control plates were subtracted from those testing direct interactionsamong ISLT proteins and LIP5(1–159), CHMP1, or SKD1. Between 250 and 400colonies were counted in each case. Two independent replicates were used forquantification. For each tested interaction, we also detected the activity of thelacZ reporter gene by a colony filter lift assay using X-gal (5-bromo-4-chloro-3-N-dolyl-b-d-galactoside) as a substrate of b-galactosidase.

Phylogenetic Analysis of ISTL1 Protein Sequences

The IST1 and ISTL1 protein sequences were obtained from the NationalCenter for Biotechnology Information protein database and aligned usingT-Coffee (http://www.tcoffee.org/Projects/tcoffee/; Notredame et al., 2000).Maximum likelihood-based inference and bootstrap analyses with 10,000 rep-licates to estimate clade support were performed with MEGA6 (http://www.megasoftware.net/; Tamura et al., 2013). Sequences from Trypanosoma andLeishmania were chosen as outgroup.

Accession Numbers

ISTL1 At1g34220; ISTL2 At1g25420; ISTL3 At4g35730; ISTL4 At4g29440;ISTL5 At2g19710; ISTL6 At1g13340; ISTL7 At4g32350; ISTL8 At1g79910; ISTL9At1g52315; ISTL10 At2g14830; ISTL11 At3g15490; ISTL12 At1g51900; LIP5AT4G26750.

Supplemental Data

The following supplemental materials are available.

Supplemental Figure S1. Twenty-six-day-old lip5-1 and lip5-2 plants withreduced rosette size compared to wild-type Col-0.

Supplemental Figure S2. PIN2-GFP and PIN3-GFP polarization in wild-type and lip5-1 roots.

Supplemental Figure S3. Root growth rate in lip5-1 mutant and wild-typeseedlings.

Supplemental Figure S4. Normal internalization of FM4-64 in lip5 mu-tants.

Supplemental Figure S5. Expression patterns of ISTL1–6 genes in Arabi-dopsis.

Supplemental Table S1. Primer sequences.

ACKNOWLEDGMENTS

We thank Li Huey Yeun for preparing vectors for the Y2H assays, JanicePennington for preparation of samples by immunolocalization, Brendan Vorpahland Michael Baumgartner for their assistance in growing plants and preparingmaterial, the Arabidopsis Biological Resource Center for providing plant mate-rials, and Sarah Swanson (Newcomb Imaging Centre, University of Wisconsin-Madison) for her assistance with confocal imaging.

Received February 17, 2016; accepted March 15, 2016; published March 16,2016.

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