fig4: Shr3p prevents AAP aggregation. (A) Extracts from isogenic SHR3 wild-type (CAY28) and shr3Δ 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.

Mentions: AAPs insert into the ER membrane with the correct topology independently of Shr3p (Gilstring and Ljungdahl, 2000). Thus, the presence of Shr3p is not essential for the topogenic signals in AAPs to be properly interpreted by the Sec61 translocon. Although AAPs attain their correct topology, our cross-linking studies suggested that AAPs require Shr3p to fold correctly. We sought an independent experimental strategy to test this prediction and considered the possibility that AAPs may aggregate in the absence of Shr3p. The migration of Gap1p in polyacrylamide gels under nondenaturing conditions was examined; membrane preparations were partially solubilized using the nonionic detergent dodecyl-β-d-maltopyranoside (DM) and resolved by BN-PAGE (Schägger et al., 1994). Gap1p present in SHR3 wild-type membranes migrated as a diffuse band of monomers (Fig. 4 A, lanes 1 and 3). In contrast, the majority of Gap1p in membranes of shr3Δ mutant cells migrated as a high molecular weight smear (Fig. 4 A, lanes 2 and 4). Similar results were obtained when the migration of a second AAP, Agp1p (Iraqui et al., 1999), was examined (Fig. 4 B). These results, consistent with a chaperone-like function, indicate that in the absence of Shr3p, AAPs aggregate into high molecular weight complexes.

Kota J, Ljungdahl PO - J. Cell Biol. (2004)

Bottom Line: Also, we show that the integral ER proteins, Gsf2p, Pho86p, and Chs7p, function similarly to Shr3p.In cells individually lacking one of these components only their cognate substrates, hexose transporters, phosphate transporters, and chitin synthase-III, respectively, aggregate and consequently fail to exit the ER membrane.These findings indicate that polytopic membrane proteins depend on specialized membrane-localized chaperones to prevent inappropriate interactions between membrane-spanning segments as they insert and fold in the lipid bilayer of the ER membrane.

Affiliation: Ludwig Institute for Cancer Research, S-17177 Stockholm, Sweden.

Abstract: The integral endoplasmic reticulum (ER) membrane protein Shr3p is required for proper plasma membrane localization of amino acid permeases (AAPs) in yeast. In the absence of Shr3p AAPs are uniquely retained in the ER with each of their twelve membrane-spanning segments correctly inserted in the membrane. Here, we show that the membrane domain of Shr3p specifically prevents AAPs from aggregating, and thus, plays a critical role in assisting AAPs to fold and correctly attain tertiary structures required for ER exit. Also, we show that the integral ER proteins, Gsf2p, Pho86p, and Chs7p, function similarly to Shr3p. In cells individually lacking one of these components only their cognate substrates, hexose transporters, phosphate transporters, and chitin synthase-III, respectively, aggregate and consequently fail to exit the ER membrane. These findings indicate that polytopic membrane proteins depend on specialized membrane-localized chaperones to prevent inappropriate interactions between membrane-spanning segments as they insert and fold in the lipid bilayer of the ER membrane.