Gap1 requires Shr3 for proper intracellular localization. Cell lysates prepared from SHR3 (PLY127) and shr3Δ6 (FGY212) cells grown in SUD were fractionated on 12–60% step sucrose gradients. Proteins within fractions were separated by SDS-PAGE and analyzed by immunoblotting. The antibodies recognizing marker proteins Pma1 (100 kD), Wbp1 (49 kD), Kex2 (90 kD), and Dap2 (93 kD) were used to identify fractions containing PM, ER, Golgi, and vacuolar proteins, respectively. The asterisks indicate nonspecific immunoreactive bands unrelated to Gap1.

Gap1 can be split into N- and C-terminal portions that assemble to form a functional permease in an Shr3-dependent manner. (A) Schematic representation of the membrane topology of Gap1, which is composed of 602 amino acid residues. The gray boxes depict the 12 membrane-spanning segments (I–XII); the numbers within boxes refer to the amino acid residues at the beginning and end of each membrane-spanning segment. The minus and plus symbols represent the positions of the charged amino acid residues in membrane-spanning segments III, V, VI, VII, and XII. (B) Illustration of full-length Gap1 and truncated gap1 constructs. The gray boxes represent the 12 transmembrane segments, the amino acid residues included in each fragment are indicated, and asterisks indicate the insertion of a methionine residue to enable expression of C-terminal fragments. (C) Serial dilutions of cell suspensions of strain FGY15 (gap1Δ) carrying vector controls (VC; pRS316 and pRS317), or expressing full-length Gap1 (pPL247 and pRS317) or truncated gap1 constructs were prepared—gap1 TM1-5 (pJK97 and pRS317), gap1 TM1-6 (pJK96 and pRS317), gap1 TM6-12 (pJK99 and pRS316), or gap1 TM7-12 (pJK98 and pRS316), respectively. Aliquots of each dilution were applied to SAD or SAD supplemented with d-histidine (0.15%). (D) Serial dilutions of strain FGY15 (SHR3 gap1Δ) or FGY135 (shr3Δ gap1Δ) carrying vector controls or expressing full-length Gap1 or coexpressing matched pairs of truncated N- and C-terminal gap1 constructs gap1 TM1-5 (pJK97) and gap1 TM6-12 (pJK99); gap1 TM1-6 (pJK96) and gap1 TM7-12 (pJK98); or gap1 TM1-5 (pJK97) and gap1 TM6-12MYC (pJK100), as indicated, were applied to SAD or SAD with d-histidine (0.15%). Plates were incubated at 30°C for 2.5 d and photographed.

Shr3 prevents aggregation of assembled split Gap1 constructs. Extracts from strains FGY15 (SHR3 gap1Δ) and FGY135 (shr3Δ gap1Δ) expressing full-length Gap1 (pPL247 and pRS317) (A), coexpressing gap1 TM1-5 (pJK97) and gap1 6-12 (pJK99) (B), or coexpressing gap1 TM1-5 (pJK97) and gap1 6-12myc (pJK100) (C and D) were prepared and solubilized with DM at the indicated concentrations (μg DM μg⁻¹ protein). Solubilized proteins were separated by BN-PAGE on gradient gels and immunoblotted with anti–NT-Gap1 or c-myc antibodies as indicated. An aliquot of the extracts containing the truncated gap1 constructs (10 μg) was analyzed by SDS-PAGE and immunoblotted with anti–NT-Gap1 (B and C, bottom) or with anti–c-myc antibody (D, bottom). The asterisks indicate a nonspecific immunoreactive band unrelated to Gap1 that fortuitously serves as a loading control.

Temporal requirement of Shr3 in the assembly of split Gap1. Extracts from strains FGY15 (SHR3 gap1Δ) and FGY135 (shr3Δ gap1Δ) expressing gap1 TM1-5 (pJK97 and pRS317) (A) or gap1 TM6-12myc (pJK100 and pRS316) (B) were prepared and solubilized with DM at the indicated concentrations (μg DM μg⁻¹ protein). Solubilized proteins were separated by BN-PAGE on gradient gels and immunoblotted with anti–NT-Gap1 or c-myc antibodies as indicated. An aliquot of the extracts containing the truncated gap1 constructs (10 μg) were analyzed by SDS-PAGE and immunoblotted with anti–NT-Gap1 (A; bottom) or with anti–c-myc antibody (B; bottom).

Gap1 is degraded by ERAD in cells lacking SHR3. Pulse-chase analysis of Gap1 degradation in SHR3 (A and C) and shr3Δ-null mutant cells (B and D). SAD-grown cells were labeled with [³⁵S]methionine for 30 min and chased by the addition of excess unlabeled methionine and cysteine. Aliquots of cells were harvested at times indicated, and Gap1 was immunoprecipitated from cell lysates (see Materials and methods). Immunoprecipitated proteins were separated by SDS-PAGE, and the amount of Gap1-associated radioactivity in samples was quantitated by phosphorimaging. The percentage of radioactivity remaining at each time point from at least two independent experiments is plotted. (A and B) Analysis of Pep4-dependent degradation. In A, solid circles indicate SHR3 (FGY127) and open squares indicate pep4Δ (FGY214). In B, solid circles indicate shr3Δ (FGY212) strains and open squares indicate shr3Δ pep4Δ (FGY219). (C and D) Analysis of the E2 ubiquitin ligase requirement. Strains in C are indicated as follows: solid circles, SHR3 (FGY127); open circles, ubc6Δ (FGY205); solid diamonds, ubc7Δ (FGY206); open squares, ubc6Δ ubc7Δ (JKY36). Strains in D are indicated as follows: solid circles, shr3Δ (FGY212); open circles, shr3Δ ubc6Δ (FGY209); solid diamonds, shr3Δ ubc7Δ (FGY210); open squares, shr3Δ ubc6Δ ubc7Δ (JKY37).

Doa10- and Hrd1-dependent pathways redundantly target Gap1 aggregates for ERAD in shr3Δ mutant cells. Pulse-chase analysis of Gap1 degradation was performed as in Fig. 4. The percentage of Gap1 remaining in each fraction from two or more independent experiments is plotted. The graph presents an analysis of Doa10- and Hrd1-dependent degradative pathways. Strains are indicated as follows: solid circles, shr3Δ (FGY212); open circles, shr3Δ doa10Δ (JKY29); solid diamonds, shr3Δ hrd1Δ (FGY257); open squares, shr3Δ hrd1Δ doa10Δ (JKY39).

Agp1 is degraded similar to Gap1 in shr3Δ mutant cells. SHR3 and shr3Δ strains carrying plasmid pJK60 (STP1Δ131; Andréasson and Ljungdahl, 2002) to constitutively induce AGP1 expression were grown in SAD. Cycloheximide was added and lysates were prepared at the times indicated. Proteins, resolved by SDS-PAGE, were analyzed by immunoblotting using polyclonal rabbit anti-Agp1 (1:10,000). Immunoreactive bands were visualized using chemiluminescence detection reagents, and the chemiluminescent signals were quantitated. The percentage of Agp1 remaining in each fraction from two or more independent experiments is plotted. (A) Analysis of Agp1 degradation in SHR3 cells. Solid circles, SHR3 (FGY127); open circles, pep4Δ (FGY214); open squares, ubc6Δ ubc7Δ (JKY36). (B) Analysis of Agp1 degradation in shr3Δ cells. Solid circles, shr3Δ (FGY212); open circles, shr3Δ pep4Δ (FGY219); open squares, shr3Δ ubc6Δ ubc7Δ (JKY37); solid diamonds, shr3Δ hrd1Δ doa10Δ (JKY39).

Mutations that impair ERAD partially suppress shr3-null mutant phenotypes. (A) Growth characteristics of SHR3 (PLY127), shr3Δ (FGY212), shr3Δ ubc6Δ (FGY209), shr3Δ ubc7Δ (FGY210), shr3Δ ubc6Δ ubc7Δ (JKY37), shr3Δ hrd1Δ (FGY257), shr3Δ doa10Δ (JKY29), and shr3Δ doa10 hrd1Δ (JKY39) strains. Cells were resuspended in water to an equal density and 10-fold dilutions were prepared. An aliquot from each dilution was applied to SD SAD containing and d-his (0.15%) to monitor Gap1 activity. (B) Growth of the same strains as in A transformed with pJK60 (STP1Δ131) to constitutively induce SPS (Ssy1p-Ptr3p-Ssy5p) sensor–regulated AGP1 and GNP1 expression (Andréasson and Ljungdahl, 2002). Aliquots of cell suspensions were applied to SD and SD containing AzC (SD + AzC) to monitor Agp1 and Gnp1 activity (Andréasson et al., 2004). The SD media contained leucine to induce SPS sensor–regulated AAPs. Plates were incubated at 30°C for 3 d and photographed. (C) Extracts from strain PLY127 (SHR3), FGY212 (shr3Δ), JKY37 (shr3Δ ubc6Δ ubc7Δ), and JKY39 (shr3Δ doa10 hrd1Δ) grown in SAD were prepared and solubilized with DM (1.25 μg DM μg⁻¹ protein). Solubilized proteins were separated by BN-PAGE and immunoblotted with anti–NT-Gap1 antibody.