Rer1p, a retrieval receptor for ER membrane proteins, recognizes transmembrane domains in multiple modes

Abstract

The yeast Golgi membrane protein Rer1p is required for the retrieval of various endoplasmic reticulum (ER) membrane proteins such as Sec12p and Sec71p to the ER. We demonstrate here that the transmembrane domain (TMD) of Sec71p, a type-III membrane protein, contains an ER localization signal, which is required for physical recognition by Rer1p. The Sec71TMD-GFP fusion protein is efficiently retrieved to the ER by Rer1p. The structural feature of this TMD signal turns out to be the spatial location of polar residues flanking the highly hydrophobic core sequence but not the whole length of the TMD. On the Rer1p side, Tyr152 residue in the 4th TMD is important for the recognition of Sec12p but not Sec71p, suggesting that Rer1p interacts with its ligands at least in two modes. Sec71TMD-GFP expressed in the Deltarer1 mutant cells is mislocalized from the ER to the lumen of vacuoles via the multivesicular body (MVB) sorting pathway. In this case, not only the presence of polar residues in the Sec71TMD but also the length of the TMD is critical for the MVB sorting. Thus, the Rer1p-dependent ER retrieval and the MVB sorting in late endosomes both watch polar residues in the TMD but in a different manner.

Figure 1.

Rer1p-dependent ER localization of GFP-Sec12p and Sec71p-GFP. (a) Subcellular localization of GFP-Sec12p and Sec71p-GFP. The wild-type (SNY9) and Δrer1 (SKY7) cells were transformed with a single-copy plasmid containing GFP-SEC12 (TDH3 promoter) or SEC71-GFP (SEC71 promoter) and subjected to confocal laser scanning microscopy. Bright ring-shaped structures are nuclear envelopes, a part of the ER, and fuzzy large organelles seen in the Δrer1 mutant are vacuoles. (b) Immunoblotting analysis of GFP-Sec12p and Sec71p-GFP. Wild-type (SNY9) and Δrer1 (SKY7) cells expressing GFP-Sec12p under the TDH3 promoter or Sec71p-GFP under the own promoter on a single-copy (CEN) or multicopy (2 μ) plasmid were grown at 20°C. Cell extracts were subjected to immunoblotting with the anti-GFP antibody. Open arrowhead, GFP-Sec12p; closed arrowhead, Sec71p-GFP; asterisk, degradation product. (c) Physical interaction between Rer1p and Sec71p. Cell lysates of the Δrer1 Δpep4 strain (SKY42) expressing Rer1-3HAp on a multicopy plasmid and Sec71p-GFP on a single-copy (CEN) or multicopy (2 μ) plasmid were subjected to chemical cross-linking with DSP. The immunoprecipitates with the anti-GFP antibody were examined by immunoblotting with anti-GFP and anti-HA antibodies.

Figure 2.

The TMD of Sec71p contains a signal for ER localization. Various chimeras between Mfα1p, Wbp1p, and Sec71p were constructed as depicted on the left and expressed in the wild-type (SNY9), Δrer1 (SKY7), and ret1-1 (SKY27) cells. The transformants were examined for α-factor secretion by the halo assay. Note that a smaller halo indicates less secretion of α-factor, in other words, good ER localization of the chimeric protein. MW71WS (arrowhead) shows good ER localization in the wild-type but not in the Δrer1 or ret1-1 mutant.

Figure 3.

The TMD of Sec71p is sufficient for interaction with Rer1p and for ER localization. (a) The Sec71TMD and its neighboring residues (35 amino acids) were fused to the NH₂-terminus of GFP. The TMD region is underlined. This Sec71TMD-GFP fusion was expressed in the wild-type (SNY9), Δrer1 (SKY7), and Δrer1 Δvps27 (SKY80) cells and examined by confocal laser scanning microscopy. Fluorescence (A, C, and E) and Nomarski (B, D, and F) images are shown. (b) Physical interaction between Rer1p and the Sec71TMD. Lysates were prepared from Δrer1 Δpep4 cells (SKY42) coexpressing Rer1-3HAp on a multicopy plasmid and Sec71-GFP or Sec71TMD-GFP on a single-copy plasmid and incubated with or without DSP. The immunoprecipitates with the anti-GFP antibody were subjected to immunoblotting with anti-GFP and anti-HA antibodies.

Figure 4.

Mutational analysis of the Sec71TMD. In the MW71WS chimera of Figure 2, a variety of mutations were introduced into the TMD region. Putative TMDs are underlined. In the control (top), the highly hydrophobic region (HHR) and the flanking polar residues are marked with a box and asterisks, respectively. Sequences of the mutants constructed are shown below. These constructs were expressed in the wild-type (SNY9) and Δrer1 (SKY7) cells. Eight independent transformants were subjected to the halo assay and the secreted α-factor was quantified as described in MATERIALS AND METHODS. The ratio of the amount of secreted α-factor from the wild-type cells to that from the Δrer1 cells are shown on the right. Smaller numbers indicate better Rer1p-dependent ER localization.

Figure 5.

Localization of GFP-fused Sec71TMD mutants. (a) Amino acid sequences of the TMD mutants. Predicted TMD regions are underlined. (b) Effects of mutations of the polar residues. LS, YL, and SL mutants were expressed in the wild-type (SNY9) and Δrer1 (SKY7) cells and examined by confocal laser scanning microscopy. Fluorescence (left panel in the pair) and Nomarski (right panel in the pair) images are shown. Bar, 5 μm.

Figure 6.

Effects of the length of the TMD on the localization of Sec71TMD-GFP. Control and leucine insertion mutants (1L, 2L, 3L, 4L, and 2LΔLV; a) were expressed in the wild-type (SNY9) and Δrer1 (SKY7) cells and observed by confocal microscopy (b). Fluorescence (left panel in the pair) and Nomarski (right panel in the pair) images are shown. Bar, 5 μm.

Figure 7.

Mutational analysis on the Rer1p side. (a) Schematic structure of Rer1p. Polar residues are colored: red, acidic; blue, basic; green, noncharged. Ten noncharged polar amino acid residues in or near the four TMDs (shown numbered) were selected and mutated. (b) Subcellular localization of Rer1p mutants. The polar residues marked in panel a were individually mutated to leucine in the GFP-Rer1p construct and were analyzed for localization in Δrer1 cells (SKY7) by confocal microscopy. Rer1ΔC is the truncation mutant that lacks the COOH-terminal 25 residues. Bar, 5 μm.

Figure 8.

Differential modes of interaction between Rer1p and its ligands. The Rer1pY152L mutant can recognize the Sec71TMD but not the Sec12TMD. (a and b) GFP-Rer1p and its TMD mutants complement Δrer1 to different degrees. The Δrer1 cells (SKY75) expressing either Sec12p-Mfα1p (a) or Mfα1p-Sec71p (b) were transformed with GFP-RER1 or its mutants. The transformants were examined for the α-factor secretion by the halo assay. Note that the Y152L mutant forms a large halo from Sec12-Mfα1p but not from Mfα1-Sec71p. (c) Reduced interaction between Rer1Y152L and the Sec12TMD. The Δdap2 cells (SMY22–10B) coexpressing Rer1-3HAp or Rer1Y152L-3HAp and Dap2p or DSD (Dap2-Sec12-Dap2 chimera) were subjected to cross-linking with DSP. The immunoprecipitates with anti-Dap2p or anti-HA antibodies were further analyzed by immunoblotting again with anti-Dap2p and anti-HA antibodies. (d) Rer1Y152L is normal for the interaction with the Sec71 TMD. Lysates were prepared from the Δrer1 Δpep4 cells (SKY42) coexpressing Sec71TMD-GFP and Rer1-3HAp or Rer1Y152L-3HA and incubated with or without DSP. The immunoprecipitates with the anti-GFP antibody were subjected to immunoblotting with anti-GFP or anti-HA antibodies.