Figure 3

Sec26p mutant allele creation. A. Bar chart showing the relative location and number of each mutated amino acid position identified in the ts allele set created by random mutagenesis. Superimposed is the % identity of each residue derived from a sequence alignment of βCOP sequences in Clustal v1.83. For reference, a grid indicates the positions of amino acid residues that are aligned (according to Figure 1) with solvent exposed residues of γCOP, located within 3Å distance of the FxxxW motif, and identified as a minimal set of functionally important residues through GLO3 interactions. B. Expression of constructs revealed with anti-Sec26p appendage domain antibody. Whole cell lysates were prepared from (Lane 1) wild type cells expressing endogenous Sec26p (109 kDa), (Lane 2) cells expressing GFP-Sec26p as the only copy of Sec26p (136 kDa, pRC2798), (Lane 3) cells over-expressing Sec26p under control of the copper-inducible promoter (P _CUP1 ) (42.2 kDa, pRC2630a), or (Lane 4) cells expressing both wild type levels of Sec26p plus over-expressed Sec26p appendage under control of the copper-inducible promoter (P _CUP1 ) (pRC2629b). C. Invertase secretion in sec26^tsmutant cells. Several mutant alleles were chosen and invertase secretion was measured after a one hour shift to the restrictive temperature. The graph shows % secretion relative to wild type and an alternative COPI ts mutant, the deletion of sec28 (εCOP subunit). Data plotted is the average of three experiments and confidence intervals shown with error bars. D. Sec26p allele expression levels. Cellular lysates prepared from each of the sec26^tsstrains after incubation at 40°C for 1 h to assess Sec26p expression levels at the restrictive temperature. Blots were subsequently probed with an anti-Elp1p (~150 kDa) antibody [70] as a loading control.