Ykt6 functionally overlaps with vacuolar and exocytic R-SNAREs in the yeast Saccharomyces cerevisiae

The soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex forms a 4-helix coiled-coil bundle consisting of 16 layers of interacting side chains upon membrane fusion. The central layer (layer 0) is highly conserved and comprises three glutamines (Q) and one arginine (R), and thus SNAREs are classified into Qa-, Qb-, Qc-, and R-SNAREs. Homotypic vacuolar fusion in Saccharomyces cerevisiae requires the SNAREs Vam3 (Qa), Vti1 (Qb), Vam7 (Qc), and Nyv1 (R). However, the yeast strain lacking NYV1 (nyv1Δ) shows no vacuole fragmentation, whereas the vam3Δ and vam7Δ strains display fragmented vacuoles. Here, we provide genetic evidence that the R-SNAREs Ykt6 and Nyv1 are functionally redundant in vacuole homotypic fusion in vivo using a newly isolated ykt6 mutant. We observed the ykt6-104 mutant showed no defect in vacuole morphology, but the ykt6-104 nyv1Δ double mutant had highly fragmented vacuoles. Furthermore, we show the defect in homotypic vacuole fusion caused by the vam7-Q284R mutation was compensated by the nyv1-R192Q or ykt6-R165Q mutations, which maintained the 3Q:1R ratio in the layer 0 of the SNARE complex, indicating that Nyv1 is exchangeable with Ykt6 in the vacuole SNARE complex. Unexpectedly, we found Ykt6 assembled with exocytic Q-SNAREs when the intrinsic exocytic R-SNAREs Snc1 and its paralog Snc2 lose their ability to assemble into the exocytic SNARE complex. These results suggest that Ykt6 may serve as a backup when other R-SNAREs become dysfunctional and that this flexible assembly of SNARE complexes may help cells maintain the robustness of the vesicular transport network.


Nyv1 plasmids
NYV1 was PCR-amplified with the oligonucleotides AK3 and AK4, followed by digestion with BamHI and EagI to ligate pRS413 to generate pRS413-Nyv1.The plasmid pRS413-Nyv1-R192Q was generated by gap repair cloning using PCR products and vector DNAs, as shown in Table S5.The plasmids pRS413-3FLAG-Nyv1 and pRS413-3FLAG-Nyv1-R192Q were generated by seamless cloning using PCR products and vector DNAs, as shown in Table S6.

Sec22 plasmids
Plasmids pRS416-Sec22 and pRS416-Sec22-R157Q were generated via the gap repair cloning method using PCR products and vector DNAs, as shown in Table S5.These plasmids were digested with XbaI/XhoI, and the resulting Sec22 and Sec22-R157Q fragments were ligated into pRS413 to generate pRS413-Sec22 and pRS413-Sec22-R157Q, respectively.

Snc1 plasmids
The SNC1 gene fragment was PCR-amplified with oligonucleotides AK25 and AK26 and digested with BamHI and HindIII.The resulting BamHI/HindIII fragment was ligated into the pRS425 vector to generate pRS425-Snc1.The SacI/SalI fragment of pRS425-Snc1 was subcloned into pRS413 to generate pRS413-Snc1.pRS413-Snc1-R53Q was constructed via the gap repair cloning method using PCR products amplified with the primers and DNA templates listed in Table S5.

Figure S1 .Figure S2 .
Figure S1.The vam7-Q284R strain shows a temperature-sensitive phenotype in terms of vacuole morphology.The vam7∆ cells harboring pRS415-Vam7-Q284R and pRS413 were grown in SC−His/Leu medium to the early log phase at 23 °C or 37 °C.The cells were stained with CMAC at the indicated temperatures and observed under a microscope.Scale bar: 5 µm.CMAC, 7-amino-4chloromethylcoumarin; SC, synthetic complete.

Figure S3 .
Figure S3.Analysis of physical interactions of Vam7 with Ykt6, by co-immunoprecipitation experiments.Total lysate from the WT (BY4742) and nyv1∆ strains overexpressing VAM3, VTI1, and 3FLAG−YKT6 from a single multicopy plasmid (2 μm plasmid) and either expressing GFP or GFP−Vam7 were used for immunoprecipitation with anti-GFP antibody, and the immunoprecipitates were analyzed by Western blot with anti-FLAG, anti-GFP, and anti-Pgk1 antibodies.Closed circles indicate plasmids retained in the cells used for analyses.

Figure S5 .
Figure S5.Analysis of physical interactions of Sso1 with Snc2 and Ykt6, by coimmunoprecipitation experiments.A, Total lysate from the WT cells (BY4742) expressing either 3FLAG−Snc2 or 3FLAG−Ykt6 from low-copy plasmids and either expressing GFP or GFP−Sso1 were used for immunoprecipitation with anti-GFP antibody, and the immunoprecipitates were analyzed by Western blot with anti-FLAG, anti-GFP, and anti-Pgk1 antibodies.Closed circles indicate plasmids retained in the cells used for analyses.B, Total lysate from the WT strains expressing either

Table S1 .
Yeast strains used in this study.

Table S2 .
Oligonucleotides and template DNA sequences used for gene disruptions, gene tagging, and promoter replacement in this study.

Table S3 .
Plasmids used in this study.

Table S4 .
Oligonucleotide primers used for plasmid constructions.

Table S5 .
Nucleic acids used for plasmid construction via in vivo assembly in yeast cells.

Table S6 .
Plasmid construction by seamless cloning.