It has been documented that certain TPR domains interact with the PTIEEVD sequence at the C-terminus of Hsp70 (Liu et al 1999
; Wu et al 2001
). The structure of the TPR domain of Hop cocrystallized with the C-terminal peptide of Hsp70 has been determined (Scheufler et al 2000
), and residues at specific locations within this TPR domain responsible for interacting with the Asp residue of Hsp70 have been unequivocally defined. These residues are conserved among different Hsp70-interacting TPR domains, so using this conservation as a criterion, we searched the genome of S. cerevisiae
to identify putative Ssa1-interacting proteins that contain the TPR domain. Among the proteins obtained, only Sgt2 (Yor007c) was not shown previously to interact with Ssa1, Hsc82, or Hsp104. The alignment of the TPR domain of Sgt2 with that of Hop is shown in . Therefore, the first question considered in this study was whether Sgt2 might interact with Ssa1. Yeast lysates were prepared and immunoprecipitation was performed with anti-Sgt2 antibodies. The precipitated proteins were resolved by sodium dodecyl sulfate (SDS) gel electrophoresis. Subsequently, antibodies prepared against Ssa1 were used to determine whether Ssa1 could co-immunoprecipitate with Sgt2. The results shown in indicated that Ssa1/Ssa2 indeed was associated with Sgt2, albeit only a small fraction of Ssa proteins coprecipitated with Sgt2. Because SGT (putative vertebrate homologue of Sgt2) is known to interact with both Hsc70 and Hsp90, we then examined whether Hsc82 (yeast Hsp90) might also coprecipitate with Sgt2. In addition, we investigated whether Hsp104 might be associated with Sgt2 because not only is the C-terminal sequence of Hsp104 (DDID) similar to those of Ssa proteins and Hsc82 (EEVD), but also Hsc82 and Hsp104 are known to associate with some other TPR-containing proteins found in our initial database search, including Tom70, Cns1p, Sti1p, and Cpr7 (Abbas-Terki et al 2001
; Tesic et al 2003
; Young et al 2003
). Indeed, these 2 molecular chaperones coprecipitated with Sgt2 (). Conceivably, Sgt2 had the capacity to associate with Ssa proteins, Hsc82, and Hsp104.
Fig 1. Sequence comparison of the tetratricopeptide repeat (TPR) domains. The amino acid sequence of the Hsc70-interacting TPR domain (TPR1) of Hop is aligned with the TPR domain of Sgt2. The locations of the 2 helices in each repeat are shown, and the residues (more ...)
Fig 2. Co-immunoprecipitation of chaperones with Sgt2. Yeast lysates were incubated with anti-Sgt2 antibodies, and the proteins bound were then resolved by sodium dodecyl sulfate gel electrophoresis for immunoblotting analysis with the antibodies against various (more ...)
We then asked whether SGT2 might interact genetically with members of SSA molecular chaperones. We therefore generated cells with SSA1, SSA2, and SGT2 deleted with several different ssa1Δssa2Δ isolates and examined the growth phenotype of the mutant. In most cases, the growth of the triple mutant (ssa1Δssa2Δsgt2Δ) appeared identical to the double mutant (ssa1Δssa2Δ). Moreover, simultaneous deletions of SGT2 with HSC82 or HSP104 showed no synthetic lethality (data not shown).
interact physically and genetically with YDJ1
(Becker et al 1996
), we wondered whether SGT2
might interact with YDJ1
, and we first investigated whether or not this interaction might be genetic. Deletion of SGT2
alone in yeast cells had no effect on its growth (not shown). We then deleted SGT2
Δ cells and examined the phenotype of the double mutant. As shown in , under normal growth conditions, deletion of SGT2
did not have a significant effect on the growth of the ydj1
Δ mutant either. However, if the mutants were exposed to a nonpermissive temperature (35°C) for 2 days and then shifted back to a permissive temperature, ydj1
Δ cells remained viable but the growth of ydj1
Δ cells was severely retarded (). The double mutant thus appeared more problematic to thermal stress. We therefore investigated whether other type of stress might also have an effect on the growth of ydj1
Δ cells. Hence, the cells were allowed to grow on plates containing formamide, and the phenotypes were then examined. Indeed, in the presence of formamide, the growth of the double mutant was much worse than that of ydj1
Δ (). Thus, a genetic interaction between SGT2
was observed after the cells were placed under stress. We next asked whether or not Ydj1 associated with Sgt2. clearly demonstrates that a significant fraction of Ydj1 did indeed coprecipitate with Sgt2. To further investigate whether Sgt2 interacts physically with Ydj1, we performed a pull-down assay with purified recombinant GST-Ydj1 and Sgt2, but the result was negative (data not shown).
Fig 3. Genetic interaction between SGT2 and YDJ1. Liquid cultures (optical density at 600 nm [OD600] ≈ 4 to 6) were diluted to OD600 = 0.1 with sterile water. Three microliters of cell suspension were then spotted on yeast extract/peptone/dextrose (more ...)
It was intriguing as to why a large fraction of Ydj1 coprecipitated with Sgt2 (), even though these 2 proteins did not appear to interact physically with each other (pull-down assays in previous paragraph). One of the possibilities was that the coprecipitation of Ydj1 with Sgt2 was mediated by some other unidentified protein or proteins. Examination of the documented protein-protein interaction in the SGD database revealed that Mdy2 is one of the proteins that interacts with Sgt2. Mdy2 contains a ubiquitin-like domain; and deletion of MDY2
in S. cerevisiae
brings about a defect in nuclear migration during the mating process, resulting in a reduction in mating efficiency (Hu et al 2006
). Previously, the use of Sgt2 as bait for a yeast 2-hybrid screen, we also discovered Mdy2 as the predominant Sgt2-interacting protein (unpublished observation). Moreover, a substantial amount of Mdy2 was indeed coprecipitated with Sgt2 (), further supporting the notion that these 2 proteins interacted with each other. We subsequently identified the region in Sgt2 responsible for its interaction with Mdy2: Deletion mutants of Sgt2 were generated for yeast 2-hybrid assays. As shown in , the TPR domain and the C-terminal region of Sgt2 were not required for its interaction with Mdy2. In fact, the N-terminal fragment of Sgt2 is necessary and sufficient for interacting with Mdy2. This conclusion was in agreement with the results of the pull-down assays shown in . Here, full-length Mdy2 was fused with GST and mixed with recombinant Sgt2 and its fragments, and the polypeptides bound to GST-Mdy2 were isolated with glutathione-Sepharose. Clearly, Sgt2 physically interacted with Mdy2 (, lane 4). Indeed, the N-terminal fragment of Sgt2 was sufficient for the association of Sgt2 with Mdy2 (, lane 5).
Fig 4. The N-terminal region of Sgt2 interacts with Mdy2. (A) Yeast 2-hybrid analysis. The open reading frame of SGT2 and its deletion mutants were constructed in pAS2-1, and MDY2 was constructed in pGAD-Cx. The plasmids were transformed into yeast strain Y190 (more ...)
We next determined whether Mdy2 might interact physically with Ydj1. We first determined whether Ydj1 could be precipitated from yeast lysates by antibodies against Mdy2. The results shown in clearly demonstrated that this was the case. We then determined whether Mdy2 physically interacts with Ydj1. Therefore, we prepared GST-Mdy2 from bacteria and Ydj1 from yeast cells (), and we then incubated the proteins. Subsequently, proteins associated with GST-Mdy2 were analyzed with anti-Ydj1 antibodies to determine whether Ydj1 interacted with Mdy2. Immunoblotting was necessary because the electrophoretic mobilities of GST-Mdy2 and Ydj1 are similar, if not identical, on SDS gels. The results shown in (lanes 2 and 3) clearly demonstrated that Ydj1 was associated with Mdy2. Moreover, addition of Sgt2 in the reaction mixtures for the pull-down assay did not affect the association of Ydj1 with Mdy2 (; lanes 4 and 5), implying that the interaction between Mdy2 and Ydj1 was independent of Sgt2.
Fig 5. Association of Ydj1 with Mdy2. (A) Co-immunoprecipitation of Ydj1 with Mdy2. Yeast lysates were prepared and were incubated with resin coupled with anti-Mdy2 antibodies. The bound proteins were eluted by acid, and the eluates were subjected to immunoblot (more ...)
We next verified in vitro whether coprecipitation of Ydj1 with Sgt2 could be mediated by Mdy2. Therefore, recombinant GST-Sgt2, GST-sgt2(ΔN) (GST fusion protein of Sgt2 with its first 58 amino acids deleted), and Mdy2 were purified (). We then performed GST pull-down assays. The results shown in demonstrated that GST-Sgt2 interacted with Mdy2, but that GST-sgt2(ΔN) lost the capacity to form complexes with Mdy2. Subsequently, we incubated the GST-Sgt2 and GST-sgt2(ΔN) with purified Ydj1 in the presence and absence of Mdy2. The Ydj1 pull-down by the fusion proteins was then determined by immunoblotting analysis. As shown in , a small fraction of Ydj1 was associated with GST-Sgt2 in the absence of Mdy2 (lane 2). However, the association of Ydj1 with Sgt2 was diminished once the N-terminal region of Sgt2 was deleted (lane 3). More importantly, the association of Ydj1 with GST-Sgt2 was much greater when Mdy2 was added to the reaction mixtures (lane 5). These results indicated that Mdy2 had the capacity to mediate the association of Ydj1 with Sgt2.
Fig 6. Mdy2 effects on the interaction of Sgt2 and Ydj1. (A) Purified proteins: GST-Sgt2 (lane 1), GST-sgt2(ΔN) (lane 2), glutathione S-transferase (GST) (lane 3), and Mdy2 (lane 4) were expressed in bacteria and were purified as described in “Materials (more ...)
Now that we have shown that Mdy2 interacts with Ydj1 and Sgt2, we wanted to determine whether or not Mdy2 might interact with Ssa chaperones. We first prepared yeast lysates and performed immunoprecipitation with anti-Mdy2 antibodies. The precipitated proteins were subjected to immunoblotting with anti-Ssa1 antibodies. Our result demonstrated that little Ssa1/Ssa2 co-immunoprecipitated with Mdy2 (data not shown). Similarly, antibodies against Mdy2 did not bring down any Hsc82 or Hsp104 either (data not shown). We then generated a triple mutant ssa1Δssa2Δmdy2::hphMX4 and examined its growth phenotype. The results demonstrated that the growth of this triple mutant was indistinguishable from that of ssa1Δssa2Δ in the presence or absence of formamide (data not shown).
The next question asked was whether or not MDY2 and YDJ1 might interact genetically. We therefore generated a double mutant ydj1Δmdy2Δ yeast strain with both YDJ1 and MDY2 deleted and examined the growth phenotype of the mutants. Although the growth of mdy2Δ was identical to that of the wild type yeast (not shown), the result shown in clearly indicated that the growth of the double mutant was severely retarded compared with that of ydj1Δ. Evidently, MDY2 and YDJ1 interact genetically.
Fig 7. Genetic interaction of YDJ1 and MDY2. Mutant ydj1Δmdy2Δ was generated and verified by Western blot analysis. Equal units of cells at an optical density of 600 nm (OD600) carrying the indicated mutations were streaked onto YPD plates and (more ...)
Previously, it was shown that the deletion of MDY2
results in a reduction in the mating efficiency of S. cerevisiae
(Hu et al 2006
). The last question considered in this study was whether deletion of SGT2
Δ background might further suppress the mating efficiency of the yeast. Therefore, we mixed the mutants (MAT
α) with a tester strain (MATa
) at a ratio of 1:10 for mating. Subsequently, we measured the mating efficiency. Under these conditions, the difference in growth rate of haploid cells on mating efficiency would be minimized, and small variations in mixing and plating of the cells were not likely to affect the measurement. As shown in , 70% of the wild-type cells were mated in an hour. The mating efficiency of the mdy2
Δ strain was reduced, albeit the level of the reduction was not as dramatic as that previously reported (Hu et al 2006
). Interestingly, deletion of SGT2
also resulted in a small reduction in mating efficiency. However, deletion of SGT2
in an mdy2
Δ background did not further suppress the mating efficiency compared with the mdy2
Δ strain. On the other hand, compared with the wild-type cells, deletion of YDJ1
resulted in a 15-fold reduction in mating efficiency. In addition, deletion of both YDJ1
brought about a further 10-fold reduction in mating efficiency. Therefore, the genetic interaction between YDJ1
as determined by growth () was also revealed in the mating efficiency measurement.
Fig 8. Mutants show reduction in mating efficiency. BY4742 and various mutants with an α mating type were mixed with an a-type tester strain (TFW4272) at a ratio of 1:10, and they were allowed to mate at 30°C for an hour. The mating efficiency (more ...)