Gap1p-GFP localizes to the PM in an Shr3p-dependent manner. Strain FGY135 (shr3Δ) carrying plasmid pJOD010 (PGAL-GAP1-GFP) was cotransformed with plasmids pJK64 (SHR3), pJK65 (shr3ΔCT), or pRS317 (shr3Δ, vector without insert). Cells were grown in allantoin media with 2% raffinose and 0.2% glucose to an OD₆₀₀ of 2–3, and subsequently suspended in allantoin media with 4% galactose and 1 μg ml⁻¹ DAPI for 5 h. (A) Cells immobilized in low melting point agar (0.4%) were viewed by Nomarski optics and the GFP and DAPI fluorescence was examined using an Axiophot microscope outfitted with a CCD camera. Bar, 5 μm. (B) Extracts from each of the transformants were prepared, proteins were resolved by SDS-PAGE, and immunoblotted with monoclonal anti-GFP antibody (Roche).

Shr3p prevents AAP aggregation. (A) Extracts from isogenic SHR3 wild-type (CAY28) and shr3Δ null mutant (JKY1) strains grown in SAD at 23°C were prepared and solubilized with DM at the indicated concentrations (μg DM μg⁻¹ protein). Solubilized proteins were separated by BN-PAGE and immunoblotted with anti-Gap1p antibody. (B) Agp1p in extracts from strains PLY127 (SHR3) and FGY212 (shr3Δ), carrying plasmid pJK60 (STP1Δ131) (Andréasson and Ljungdahl, 2002) to induce AGP1 expression, were solubilized and analyzed as in A using anti-Agp1p antibodies.

Shr3p-dependent cross-linking and aggregation occurs in the ER. (A) Gap1p is retained in the ER of sec12-1 mutant strains grown at nonpermissive temperatures. Strains JKY3 (sec12-1) and JKY4 (sec12-1 shr3Δ), grown at 22°C (permissive) in ammonium-based SC to repress GAP1 expression, were harvested, washed, and resuspended in allantoin containing medium (SAD) for 2 h at 34°C (nonpermissive) to induce GAP1 expression. Extracts were fractionated on 12–60% step sucrose gradients, and proteins within fractions 1–9 were separated by SDS-PAGE and analyzed by immunoblotting. Antibodies recognizing marker proteins Pma1p, Sec61p, and Kex2p were used to identify fractions containing PM, ER, and Golgi proteins, respectively. (B and C) Gap1p retained in the ER of sec12-1 strains cross-links and aggregates in an Shr3p-dependent manner. (B) Immunoblot analysis of DSP and SANPAH treated extracts as in A with anti-Gap1p antibody. (C) Extracts as in A were solubilized with DM at the indicated concentrations (μg DM μg⁻¹ protein). Solubilized proteins were separated by BN-PAGE and immunoblotted with anti-Gap1p antibody.

Structural and functional analysis of Shr3p. (A) Schematic illustration of the predicted membrane topology of Shr3p. The NH₂ (NT) and COOH termini (CT) are predicted to be oriented toward the same side of the ER membrane. The four hydrophobic segments (I–IV) are separated by hydrophilic loops L1–L3. The suc2 topological reporter cassette (see Materials and methods) was inserted at the positions indicated; the numbers refer to the amino acid immediately preceding the individual cassette insertions. (B) The experimentally determined in vivo topology of Shr3p. Extracts from FGY212 (shr3Δ) cells carrying either plasmids (pJK40-pJK44) encoding the five fusion proteins or a control vector without insert (vector, pRS316) were treated with endoglycosidase H as indicated, and proteins were resolved by SDS-PAGE and immunoblotted with anti-Shr3p antibody. The orientation of each reporter cassette is indicated (cyt, cytosolic; lum, lumenal). (C) The hydrophobic membrane domain of Shr3p is sufficient for function. The wild-type SHR3 and the COOH-terminally truncated shr3ΔCT alleles are depicted with sequences encoding the four putative transmembrane domains (I–IV) are represented by the gray boxes. Serial dilutions (10-fold) of cell suspensions of strain FGY212 (shr3Δ) carrying a vector control (Δ, pRS316), or plasmids expressing either Shr3p (+, pPL210) or Shr3ΔCTp (ΔCT, pJK36) were applied to SD supplemented with lysine and the indicated toxic amino acid analogue. Plates were incubated at 30°C for 4 d and photographed. The abbreviations of amino acid analogues and concentrations used are as follows: Ethionine, d,l-Ethionine (60 μg ml⁻¹); f-PA, p-fluoro-d,l-phenylalanine (400 μg ml⁻¹); AzC, azetidine-2-carboxylate (500 μg ml⁻¹).

Gap1p specifically cross-links in the absence of Shr3p. (A) Extracts from isogenic SHR3 wild-type (CAY28) and shr3Δ null mutant (JKY1) strains grown in SAD at 23°C. Extracts were treated with the indicated concentration (mM) of DSP (left) or SANPAH (right). The DSP extracts were treated with DTT and extracts containing SANPAH were irradiated with UV as indicated. Proteins were resolved by SDS-PAGE, and immunoblotted with polyclonal anti-Gap1p antibody. (B) Immunoblot analysis of DSP treated extracts as in A with anti-Dpm1p and -Sec61p antibodies, and extracts from strains grown in SC with 4% glucose with anti-Hxt1p antibodies.

The membrane domain of Shr3p suffices to block AAP aggregation. Extracts prepared from strain JKY1 (shr3Δ) grown in SAD carrying a vector control (Δ), or plasmids expressing either Shr3p (+) or Shr3ΔCTp (ΔCT) were prepared. (A) Extracts were solubilized with DM at the indicated concentrations (μg DM μg⁻¹ protein). Solubilized proteins were separated by BN-PAGE and immunoblotted with anti-Gap1p antibody. (B) Proteins within extracts were analyzed by SDS-PAGE and immunoblotted with anti-Gap1p and anti-Dpm1p antibodies as indicated.

Specialized membrane-localized chaperones prevent the aggregation of specific sets of polytopic membrane proteins. (A) Whole cell extracts, prepared from isogenic GSF2 wild-type (CAY28) and gsf2Δ null mutant (JKY5) strains grown in SC (for the analysis of Hxt1p and Sec61p) or in SAD (for the analysis of Gap1p). The media were supplemented with 4% glucose to induce HXT1 expression. The extracts were treated with DSP at the concentrations (mM) and DTT as indicated. Proteins in the extracts were resolved by SDS-PAGE and immunoblotted with anti-Hxt1p, anti-Gap1p, and anti-Sec61 antibodies. (B) Pho84p expression was induced in isogenic PHO86 wild-type (CEN.PK113-5D/Pho84^myc) and pho86Δ null mutant (JKY6) strains by growth in low phosphate medium. Extracts treated with DSP and DTT as in A, proteins were resolved by SDS-PAGE, and immunoblotted with anti-Myc antibody (9E10). (C) Analysis of DSP and DTT treated extracts from isogenic CHS7 wild-type (Y1306) and chs7Δ null mutant (JAY27) strains grown in SC. Proteins resolved by SDS-PAGE were immunoblotted with anti-HA antibody.