New gain-of-function SLY1* alleles. (A) Selection used in this study. A library of SLY1* alleles was constructed by mutagenic PCR and cloned into a single-copy plasmid. The library was then transformed into a SLY1 uso1∆ strain, with USO1 provided on a balancer plasmid bearing the counterselectable URA3 marker. Ejection of pUSO1 was forced by 5-fluoroorotic acid (5-FOA). This strategy positively selects viable cells carrying dominant mutant SLY1* alleles that bypass the otherwise essential USO1 requirement. (B) Locations within Sly1 (PDB ID 1MQS) of single missense substitutions that suppress requirements for Ypt1, or for both Ypt1 and Uso1. The loop is indicated in purple, with the dashed line denoting the portion of the loop not resolved in the crystal structure. Yellow shading indicates the domain 3a helical hairpin which, by analogy to Vps33 and Munc18-1, is hypothesized to scaffold assembly of Qa- and R-SNARE trans-complexes.
Figure 1.

New gain-of-function SLY1* alleles. (A) Selection used in this study. A library of SLY1* alleles was constructed by mutagenic PCR and cloned into a single-copy plasmid. The library was then transformed into a SLY1 uso1∆ strain, with USO1 provided on a balancer plasmid bearing the counterselectable URA3 marker. Ejection of pUSO1 was forced by 5-fluoroorotic acid (5-FOA). This strategy positively selects viable cells carrying dominant mutant SLY1* alleles that bypass the otherwise essential USO1 requirement. (B) Locations within Sly1 (PDB ID 1MQS) of single missense substitutions that suppress requirements for Ypt1, or for both Ypt1 and Uso1. The loop is indicated in purple, with the dashed line denoting the portion of the loop not resolved in the crystal structure. Yellow shading indicates the domain 3a helical hairpin which, by analogy to Vps33 and Munc18-1, is hypothesized to scaffold assembly of Qa- and R-SNARE trans-complexes.

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