Characterization of Yor1-ΔF/R1116T. (A) The initial characterization of YOR1 alleles was performed using plasmid-based mutagenesis. A yor1 null strain, yor1-Δ0, was transformed with a plasmid control (pRS316), or plasmids expressing YOR1, yor1-ΔF670, or yor1-ΔF670/R1116T as indicated, and the strains were serially diluted and spotted onto YPEG media with and without 0.2 µg/mL oligomycin. The yor1-ΔF670 mutation was associated with a trafficking and pump defect that rendered it phenotypically equivalent to yor1-Δ0. However, an additional intragenic mutation, Yor1-ΔF670-R1116T, exhibited an intermediate phenotype. (B) Capture of Yor1 into ER-derived transport vesicles was measured using an in vitro vesicle budding assay that quantifies uptake of newly synthesized cargo proteins from radiolabeled permeabilized cells after addition of purified COPII proteins in the presence (+) and absence (-) of GTP. Total membranes (T) were separated from the liberated vesicles by differential centrifugation. Packaging of Yor1 into the vesicle fraction was monitored by immunoprecipitation; Sec22 is a control cargo protein that demonstrates efficient vesicle production even in the absence of packaging of the mutant forms of Yor1. Neither Yor1-ΔF nor Yor1-ΔF/R1116T were captured into COPII vesicles whereas wild-type Yor1 was packaged normally. (C) Trypsin sensitivity of Yor1 was assessed by limited proteolysis of microsomal membranes expressing wild type and mutant forms of Yor1. Increasing concentrations of trypsin were added as indicated prior to processing of membranes for immunoblot analysis. Wild-type Yor1 is cleaved to several stable bands whereas both Yor1-ΔF and Yor1-ΔF/R1116T were significantly more susceptible to proteolytic attack. (D) Cross-linking between transmembrane domains of Yor1 was measured following introduction of paired cysteine substitutions into wild-type and mutant Yor1 as indicated. Addition of increasing concentrations of cross-linker resulted in the accumulation of wild-type Yor1 in a cross-linked species with distinct gel mobility. Cross-linking of Yor1-ΔF and Yor1-ΔF/R1116T resulted in the disappearance of the non-cross-linked species and the appearance of high molecular weight aggregates, suggesting abnormal assembly of transmembrane domains in these mutants. (E) Yor1 stability was monitored by pulse-chase analysis. Cells expressing wild-type or mutant forms of Yor1 were radiolabeled for 10 min, and then chased for 180 min with non-radioactive amino acids. The amount of Yor1 present at each time point was determined by immunoprecipitation and autoradiography (top panel). The percentage of Yor1 remaining was calculated relative to the starting material at t = 0 (bottom panel). (F) Yor1 function was probed using a rhodamine-pumping assay. Yor1-Δ0/Pdr5-Δ0 cells carrying the indicated Yor1 alleles on a plasmid were loaded with the fluorescent dye, rhodamine, and the amount of fluorescence released over time into the culture supernatant was measured. The Yor1-ΔF670-R1116T mutation was associated with rhodamine extrusion intermediate between that of the wild-type Yor1 allele and either the Yor1-Δ0 or Yor1-ΔF670 mutant forms (which were functionally equivalent in this assay, as in the oligomycin resistance growth phenotype assay).