Figure 2.

Phenotypes of yeast cells containing ER-to-Golgi SNARE complexes with 2R:2Q stoichiometry depend on the positions of the arginines. (A) Cells expressing ER-to-Golgi SNARE mutations that introduce a second arginine into the SNARE complex either at position Qa or Qc are viable. Four viable spores were obtained from a heterozygous SED5 or BET1 knockout strain (strains Y21581 and Y21396), which harbors a plasmid-encoded sed5(R) (pCG32) or bet1(R) (pCG27) mutation. Sed5(R) or bet1(R) mutant cells (arrows) were resistant to Geneticin (G418⁺) as the KanMX cassette was used to replace the SED5 gene. They were Ura⁺ because they express the sed5(R) or bet1(R) mutation on plasmids carrying the URA3 marker. Replica plating also showed that the sed5(R) cells are temperature sensitive at 35°C. Unlike the sed5(R) mutation, however, the bet1(R) mutants showed no growth defect. (B) The bos1(R) mutant as the only source of BOS1 is not viable. Left, two of the tetrads obtained from a heterozygous BOS1 knockout strain (Y22689) expressing bos1(R) (pCG69). All viable spores are Geneticin sensitive, indicating that they carry the wild-type BOS1 gene. Subsequent analysis of the viable spores indicated that the bos1(R) plasmid was not lost during sporulation. However, cotransformation of bos1(R) and sec22(Q) into Y22689 yielded tetrads with four viable spores. Two spores carried the marker used for the deletion of BOS1 (right, arrows). Analysis of their auxotrophic markers demonstrated that all Geneticin-resistant spores contained both plasmids, indicating that the bos1(R) mutants require sec22(Q) for viability. (C) Introducing an additional copy of bet1(Q) raises the symmetry in the `0' layer, but it has only mild effects on the growth phenotype. Cells were grown on YEPD for 60 h (top) or 65 h (bottom). In a 2R:2Q ratio where the second arginine is contributed by sed5(R) (strain YCG74) additional expression of bet1(Q) resulted in moderate improvement of growth at 32°C. When bet1(Q) was introduced into strain YCG9 [sec22(R) + sec22(Q) bos1(R)], where the second arginine is shifted to the Qb-position, no improvement of growth was observable (bottom). (D) Two arginines at positions Qa and Qc are tolerated. Yeast cells (strain YCG103), expressing bet1(R), sec22(Q), and sed5(R) were viable but sensitive to higher temperature (37°C). The resulting ER-to-Golgi SNARE complex contained two opposing arginines at positions-Qa and -Qc. Strains were spotted on YEPD plates and grown for 48 h.