The Sly1 regulatory loop has a positive function in vivo. (A) AlphaFold2 rendering, showing the location of Sly1 loop replacement with engineered linkers (blue). Sequences of the linker insert designs, and growth phenotypes of the corresponding mutants are presented in Table S2. The domain 3a SNARE assembly template is shown in yellow. (B) The sly1∆loop mutant is temperature-sensitive for growth. Dilutions of liquid cultures were spotted as 10× serial dilutions onto YPD agar plates and incubated for 2 days at 30° or 37°C. These are knock-ins at the genomic SLY1 locus, in the Y8205 strain background used for SGA analysis. (C) Selected SGA results. Genes exhibiting synthetic interactions with sly1∆loop are shown. Scores indicate loge synthetic growth defects (red) or intergenic suppression (blue). A score of −4.6 indicates a 100× synthetic growth defect. Complete SGA results are presented in Data S1. (D) Gene Ontology Overrepresentation Test of the sly1∆loop SGA dataset. Genes with loge synthetic defect scores less than or equal to −0.5 were included in the analysis. Bars show all GO-Slim Biological Process categories with statistically significant enrichment scores (*P < 0.05; **P < 10−2; ***P < 10−6). P values were calculated using Fisher’s exact test and adjusted for multiple comparisons (Bonferroni’s correction; count = 732). Additional details are presented in Data S1. (E)SEC17 overproduction is toxic in cells expressing sly1∆loop. sly1∆ mutant cells were maintained with a counterselectable SLY1 balancer plasmid and transformed with single-copy plasmids bearing either SLY1 or sly1∆loop, as well as plasmids carrying SEC17 or sec17-FSMS (Schwartz and Merz, 2009). The balancer plasmid was ejected by plating dilutions on media with 5-FOA and growth was assayed after 2 days of growth at 30°C.
Figure 4.

The Sly1 regulatory loop has a positive function in vivo. (A) AlphaFold2 rendering, showing the location of Sly1 loop replacement with engineered linkers (blue). Sequences of the linker insert designs, and growth phenotypes of the corresponding mutants are presented in Table S2. The domain 3a SNARE assembly template is shown in yellow. (B) The sly1∆loop mutant is temperature-sensitive for growth. Dilutions of liquid cultures were spotted as 10× serial dilutions onto YPD agar plates and incubated for 2 days at 30° or 37°C. These are knock-ins at the genomic SLY1 locus, in the Y8205 strain background used for SGA analysis. (C) Selected SGA results. Genes exhibiting synthetic interactions with sly1∆loop are shown. Scores indicate loge synthetic growth defects (red) or intergenic suppression (blue). A score of −4.6 indicates a 100× synthetic growth defect. Complete SGA results are presented in Data S1. (D) Gene Ontology Overrepresentation Test of the sly1∆loop SGA dataset. Genes with loge synthetic defect scores less than or equal to −0.5 were included in the analysis. Bars show all GO-Slim Biological Process categories with statistically significant enrichment scores (*P < 0.05; **P < 10−2; ***P < 10−6). P values were calculated using Fisher’s exact test and adjusted for multiple comparisons (Bonferroni’s correction; count = 732). Additional details are presented in Data S1. (E)SEC17 overproduction is toxic in cells expressing sly1∆loop. sly1∆ mutant cells were maintained with a counterselectable SLY1 balancer plasmid and transformed with single-copy plasmids bearing either SLY1 or sly1∆loop, as well as plasmids carrying SEC17 or sec17-FSMS (Schwartz and Merz, 2009). The balancer plasmid was ejected by plating dilutions on media with 5-FOA and growth was assayed after 2 days of growth at 30°C.

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