FIGURE 1:

Domain analysis of P. pastoris Sec16. (A) Summary of the effects of Sec16 partial deletions on cell viability. Top, the UCR (residues 500–868), CCD (residues 1030–1459), a nonconserved glutamine-rich region (Q; residues 1829–1958), and the conserved CTR (residues 2392–2550). The endogenous SEC16 gene was replaced with alleles containing the indicated deletions. Right, the ability of each mutant allele to support growth. Only the deletions marked in red caused a loss of viability. (B) Differential effects of a CCD point mutation and a CCD deletion. The indicated mutations were introduced at the SEC16 locus by gene replacement in a strain expressing Sec13-GFP. Cultures were grown at 23°C, and then half of each culture was shifted to 36.5°C for 1 h. Cells were imaged by differential interference contrast and fluorescence microscopy. Scale bar, 5 μm. (C) Yeast two-hybrid analysis of Sec16-COPII interactions. The “prey” vector encoded the indicated fragments of P. pastoris Sec16, and the “bait” vector encoded the indicated full-length P. pastoris COPII coat proteins. Growth on plates lacking histidine reflects an interaction. With this system, the UCR can self-activate when used as bait, so constructs containing the UCR were tested only as prey. The other constructs gave the same results when the Sec16 fragments were used as bait and the COPII coat proteins were used as prey (unpublished data).