Introduction

Yeast Two-Hybrid Assay Demonstrates Ltv1 and Enp1 Interaction in vivoJosh Cena, Henry Lemus, Adrian Yee

Molecular Biology Lab 312

Ribosomes are important biological machines that synthesize the necessary proteins for all living organisms. Ribosome biogenesis begins in the nucleolus where ribosomal RNAs (rRNAs) bind to ribosomal proteins (r-protein) to assemble the pre-40S subunit, one of two ribosomal subunits. The pre-40S is then exported out of the nucleus through nuclear pores to the cytoplasm, where it forms a functioning ribosome by combining with the other ribosomal subunit (Figure 1).2

Figure 1. Ribosome biogenesis of the pre-40S subunit. Pre-40S nuclear export is dependent on exportin Crm1.2 Ltv1 binds the pre-40S along with Ran-GTPase and Crm1.3 Image was repurposed and edited from Johnson et al.2

We hypothesize that Ltv1 and Enp1 do interact with each other in vivo. If the hypothesis holds true then the next questions that should be addressed are, “do Ltv1 and Enp1 interact directly in vivo?” and, “what region of Ltv1 is necessary for in vivo interaction with Enp1?” We will test our hypothesis using a yeast two-hybrid assay.

Reporter Gene

RpS3

DNA-Binding Domain

Enp1 Activation Domain

DNA-Binding Sequence

Ltv1

RpS3

DNA-Binding Domain

Enp1

Activation Domain

DNA-Binding Sequence

Ltv1

Reporter Gene

Figure 3. Two models that show the possible in vivo interactions between Ltv1, Enp1, and RpS3. (Left) Ltv1 and Enp1 interaction mediated by RpS3. (Right) Direct interaction between Enp1 and Ltv1 with RpS3 bound to Ltv1.

Plasmid Promoter Terminus-AD/BD Selectable Marker

pGADCg-Ltv1 ADH1 C-AD LEU2

pGADCg-Ltv1ΔC13 ADH1 C-AD LEU2

pGADCg-Ltv1Δ56-102ΔC13 ADH1 C-AD LEU2

pGADCg-Ltv1ΔN73-ΔC13 ADH1 C-AD LEU2

pGADCg-Ltv1ΔN92ΔC13 ADH1 C-AD LEU2

pGBKT7g (Enp1 Empty Vector) ADH1 N-BD TRP1

HJA-GBK-Enp1-S (Enp1 HJA) ADH1 N-BD TRP1

Enp1-S (Enp1 BAM) ADH1 C-AD LEU2

pGADCg-Ltv1Δ202-321ΔC13 ADH1 C-AD LEU2

pGADCg-ltv1Δ322-332ΔC13 ADH1 C-AD LEU2

pGADCg-Ltv1Δ333-394ΔC13 ADH1 C-AD LEU2

pGADCg (Ltv1 Empty Vector) ADH1 C-AD LEU2

RpS3 Vector ADH1 C-AD LEU2

Strain Description

PJ69:MATa MATa trp1-901 leu2-3,112 ura3-52 his3-200 gal4Δ gal80Δ LYS2::GAL1-HIS3 GAL2-ADE2 met2::GAL7-lacZ

MethodsThe Enp1 gene containing a stop codon was amplified by PCR from yeast genomic DNA. An entry vector containing the Enp1 gene was created using a BP Gateway reaction. The entry vector was then transformed into E. coli and amplified. The entry vector was purified using plasmid DNA mini-prep. The insertion in the entry vector was identified as Enp1 using a restriction digest. Enp1 from the entry vector was inserted into destination vector pGBKT7g using an LR Gateway reaction. The resulting Enp1 expression vectors were then transformed into yeast strain PJ69:MATa along with Ltv1 expression vectors.

Figure 4. Parent vectors pGBKT7g and pGADT7g containing the GAL4 DNA-binding domain (DBD) and the GAL4 activation domain (AD) on the N-terminus.6 Ltv1 was inserted into pGADT7 to create Ltv1-AD fusion expression vectors. Enp1 was inserted into pGBKT7g to create Enp1-DBD fusion expression vectors. Yeast strain PJ69:MATa contains the HIS3 reporter gene regulated by the GAL1 UAS and the ADE2 reporter gene regulated by the GAL2 UAS.1 Image reproduced from Stellberger et al.6

Table 1. The different plasmids transformed into PJ69:MATa cells. Ltv1 deletions indicated with a Δ. The plasmids promoter, location of DNA binding and activation domains, and yeast selectable marker are shown. The genomic description of the PJ69:MATa yeast strain is shown below plasmids.1

Results

Transformed PJ69:MATa cells were streaked on LEU/TRP (LT) dropout plates. The plates were incubated at 30 °C for 4 days. Colonies from these plates were streaked for single colonies on LT plates. For interaction experiments single colonies were streaked on LT, LEU/TRP/HIS (LTH), and LEU/TRP/ADE (LTA) dropout plates. These plates were incubated at 30 °C for 72 hours and then photographed. For sterile yeast dilution assays single colonies were inoculated in 2 mL of 2% glucose LT dropout media and placed in a rotating wheel at 30 °C until the cells reached stationary phase. The optical density (OD) of the inoculated cells were measured and then diluted to 1 OD. Cells from 1 OD dilutions were used to make 1:10, 1:100, 1:1000, and 1:10,000 OD dilutions. The dilutions were then plated on LT, LTH, and LTA plates. The plates were incubated at 30 °C for 48 hours and then photographed.

Ltv1 and Enp1 interact in vivo2

1 3

2

1 3

2

1 3

LT LTH LTAFigure 5. Transformations streaked on LT, LTH, LTA plates. Cells were incubated at 30 °C for 3 days. (1) Ltv1+Enp1 HJA. (2) Ltv1+Enp1 Empty Vector. (3) Ltv1 Empty Vector+Enp1 BAM. 1, 2, and 3 all grew on the LT plate showing that the transformations were successful. Growth of 1 on the LTH and LTA plates means that the cells are expressing both HIS3 and ADE2 reporter genes. No growth for 2 and 3 on the LTH and LTA plates means the cells transformed with empty vectors did not express either reporter gene. Since 1 grows on all plates this suggests that there is possible interaction between Ltv1 and Enp1. The lack of growth of the empty vectors on LTH and LTA plates confirms that reporter gene expression is due to Ltv1 and Enp1 interaction and not autoactivation.

21

3

4

5

LT2

1

3

4

5

LTH2

1

3

4

5

LTARpS3 Does Not Mediate Ltv1 and Enp1 Interaction

Figure 6. Transformations streaked on LT, LTH, and LTA plates. Cells were incubated at 30 °C for 3 days. (1) Ltv1ΔC13+Enp1 HJA. (2) Ltv1+Enp1 HJA. (3) Ltv1+Enp1 Empty Vector. (4) Ltv1 Empty Vector+Enp1 BAM. (5) Ltv1Δ56-102ΔC13+Enp1 HJA. Since 5 does grow on LTH and LTA plates this suggests that Ltv1 and Enp1 directly interact because 5 contains an Ltv1 deletion that prevents interaction between Ltv1 and RpS3.4 If RpS3 mediates Ltv1 and Enp1 interaction then no growth would be expected for 5 on LTH and LTA plates. Because 5 grows on LTH and LTA plates it means interaction is still occurring and that their interaction is not mediated by RpS3.

Ltv1

The pre-40S subunit requires other non-ribosomal biogenesis proteins to assist with export. A new model proposed is that Ltv1 and Enp1 (biogenesis proteins) bind the pre-40s subunit by interacting with RpS3 (an important pre-40s protein) in the nucleus.3 It has been found that Ltv1, Enp1, and RpS3 form a complex in in vitro experiments, however, it is not currently known if this complex forms in vivo (Figure 2).5 This has lead us to our experimental question, “do Ltv1 and Enp1 interact in vivo?”

Figure 2. In Vitro interactions of Ltv1, Enp1, RpS3, and pre-40s. The Ltv1, Enp1, and RpS3 complex is formed and dissociates from the pre-40S when phosphorylated. RpS3 is then reinserted into the pre-40S after dephosphorylation.5 Image was repurposed and edited from Zemp et al.7

pre-40s

PhosphorylationDephosphorylation

The Region of Ltv1 Necessary for Enp1 Interaction Could not be Identified

LTALT LTH

C1

1

2

2BAM

C2

C3

5

4

3

7

8

6

1 1:101:100

1:100

01:10,000

1 1:101:100

1:100

01:10,000

1 1:101:100

1:100

01:10,000

(1) Ltv1

(5) Ltv1Δ56-102ΔC13

(4) Ltv1ΔN92ΔC13

(3) Ltv1ΔN73ΔC13

(7) Ltv1Δ322-332ΔC13

(8) Ltv1Δ333-394ΔC13

(6) Ltv1Δ202-321ΔC13

(2) Ltv1ΔC13

Figure 7. (Left) (C1) RpS3 Vector+Ltv1ΔC13. (2BAM) Ltv1+Enp1 BAM. (C2) Ltv1+Enp1 Empty Vector. (C3) Ltv1 Empty Vector+Enp1 BAM. Cell dilution assay on LT, LTH, and LTA plates. Plates were incubated at 30 °C for 2 days. Ltv1 deletions are numbered and OD dilutions are listed above. The results show no significant decrease in cell growth between different transformations. This suggests that the region necessary for Ltv1 and Enp1 interaction was not found. Transformation 6 shows less growth on all plates but this is likely due to less cells being plated. Transformation 8 shows no growth on LTA plates and growth on the other two plates. This however, cannot be interpreted as loss of interaction between Ltv1 and Enp1 since data gathered by other groups conflicts with these results. Also, 6-7 were only plated once during our experiments and would require additional tests to confirm the results. The controls were plated on separate plates but all samples were plated at the same time and grown for the same amount of time.(Right) Visual representation of the mutant Ltv1 proteins produced by expression vectors used in the yeast two-hybrid assay. The amino acid deletions are represented as black lines and the blue bars represent retained amino acids.

Ltv1 and Enp1 interact in vivo. This was seen in Figure 5 when an Ltv1+Enp1 transformation grew on LTH and LTA dropout plates while the empty vectors did not. The interaction is not mediated by RpS3 as shown in Figure 6. Ltv1Δ56-102ΔC13 cannot interact with RpS3.4 Cells transformed with the Ltv1Δ56 grew on LTH and LTA dropout plates which shows that Ltv1 and Enp1 are still interacting and that RpS3 is not mediating the interaction. The region of Ltv1 responsible for interaction with Enp1 could not be determined. Figure 7 shows no significant difference in growth between the transformations. The decreased growth of Ltv1Δ202-321ΔC13 can be attributed to less cells being plated. No growth of Ltv1Δ333-394ΔC13 deletions on LTA plates cannot be interpreted as loss of interaction since data found by other groups conflicts with these results. Further experiments are required to locate the necessary region. The region necessary interaction in Ltv1 is most likely between amino acids 102-202 or 394-450 since these regions were not tested.

Conclusions

We would like to thank Dr. Deborah Lycan, Lindy Gewin, and our TA Leah Weston for helping us with our research and guiding us in our experiments. We would also like to thank the Biology Department for giving us the necessary materials to make this project a success.

Acknowledgements

References1James, P.; Halladay, J.; Craig, E. A. (1996) Genomic Libraries and a Host Strain Designed for Highly Efficient Two-Hybrid Selection in Yeast. Gen. Soc. Am. 144(4), pp. 1425-1436.

2Johnson, A. W.; Lund, E.; Dahlberg, J. (2002) Nuclear Export of Ribosomal Subunits. Trends in Biochem. Sci. 27(11), pp. 580-585.

3Lycan, D. (2013) Molecular Biology 312 Fall 2013 Student Lab Manual. pp. 1-5.

4Lycan, D (2013) Molecular Biology Unpublished Data.

5Schafer, T.; Maco, B.; Petfalski, E.; Tollervey, D.; Bottcher, B.; Aebi, U.; Hurt, E. (2006) Hrr25-Dependent Phosphorylation State Regulates Organization of the pre-40S Subunit. Nature. 441, pp. 651-655.

6Stellberger, T; Hauser, R.; Baiker A., Pothineni, V. R.; Haas, J.; Uetz, P. (2010) Improving the Yeast Two-Hybrid System with Permutated Fusions Proteins: the Varicella Zoster Virus Interactome. Proteome Sci. 8(8), retrieved from http://www.proteomesci.com/content/8/1/8.

7Zemp, I.; Kutay, U. (2007) Minireview Nuclear Export and Cytoplasmic Maturation of Ribosomal Subunits. FEBS Letters. 581, pp. 2783-2793.