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Sorting of yeast membrane proteins into an endosome-to-Golgi pathway involves direct interaction of their cytosolic domains with Vps35p.

Nothwehr SF, Ha SA, Bruinsma P - J. Cell Biol. (2000)

Bottom Line: By genetic and biochemical means we now show that Vps35p directly associates with the cytosolic domains of cargo proteins.Furthermore, mutations in the cytosolic domains of A-ALP and another cargo protein, Vps10p, were identified that suppressed cargo-specific mutations in Vps35p but did not suppress the retrieval defects of a vps35 mutation.Suppression was shown to be due to an improvement in protein sorting at the prevacuolar compartment.

View Article: PubMed Central - PubMed

Affiliation: Division of Biological Sciences, University of Missouri, Columbia, Missouri 65211, USA. nothwehrs@missouri.edu

ABSTRACT
Resident late-Golgi membrane proteins in Saccharomyces cerevisiae are selectively retrieved from a prevacuolar-endosomal compartment, a process dependent on aromatic amino acid-based sorting determinants on their cytosolic domains. The formation of retrograde vesicles from the prevacuolar compartment and the selective recruitment of vesicular cargo are thought to be mediated by a peripheral membrane retromer protein complex. We previously described mutations in one of the retromer subunit proteins, Vps35p, which caused cargo-specific defects in retrieval. By genetic and biochemical means we now show that Vps35p directly associates with the cytosolic domains of cargo proteins. Chemical cross-linking, followed by coimmunoprecipitation, demonstrated that Vps35p interacts with the cytosolic domain of A-ALP, a model late-Golgi membrane protein, in a retrieval signal-dependent manner. Furthermore, mutations in the cytosolic domains of A-ALP and another cargo protein, Vps10p, were identified that suppressed cargo-specific mutations in Vps35p but did not suppress the retrieval defects of a vps35 mutation. Suppression was shown to be due to an improvement in protein sorting at the prevacuolar compartment. These data strongly support a model in which Vps35p acts as a "receptor" protein for recognition of the retrieval signal domains of cargo proteins during their recruitment into retrograde vesicles.

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(A) The positions of three independent mutations in the A-ALP cytoplasmic domain that specifically suppress the vps35-101 allele are shown. A schematic diagram of A-ALP is shown with the lumenal, transmembrane (TMD), and cytosolic domains indicated. The cytosolic domain residues previously shown to comprise a PVC-to-TGN retrieval signal (Nothwehr et al. 1993) are labeled with asterisks and the suppressor mutations (N88I, N88K, and N92I) are indicated with arrows. (B) The N88I mutation in the cytosolic domain of A-ALP reduces its rate of vacuolar processing in a vps35-101 strain. Yeast strains were pulse labeled with [35S]methionine/cysteine for 10 min and chased for the times indicated in minutes above the upper panel. The cells were lysed and wild-type or mutant A-ALP was immunoprecipitated and analyzed by SDS-PAGE and fluorography. The following strain–plasmid combinations were analyzed in lanes 1–4, 5–8, 9–12, 13–16, 17–20, and 21–24, respectively: SNY36-9A–pSN345, SNY110–pSN345, SNY80–pSN345, SNY36–9A–pSN346, SNY110–pSN346, and SNY80–pSN346. The positions of precursor (p) and processed mature (m) A-ALP are indicated.
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Figure 2: (A) The positions of three independent mutations in the A-ALP cytoplasmic domain that specifically suppress the vps35-101 allele are shown. A schematic diagram of A-ALP is shown with the lumenal, transmembrane (TMD), and cytosolic domains indicated. The cytosolic domain residues previously shown to comprise a PVC-to-TGN retrieval signal (Nothwehr et al. 1993) are labeled with asterisks and the suppressor mutations (N88I, N88K, and N92I) are indicated with arrows. (B) The N88I mutation in the cytosolic domain of A-ALP reduces its rate of vacuolar processing in a vps35-101 strain. Yeast strains were pulse labeled with [35S]methionine/cysteine for 10 min and chased for the times indicated in minutes above the upper panel. The cells were lysed and wild-type or mutant A-ALP was immunoprecipitated and analyzed by SDS-PAGE and fluorography. The following strain–plasmid combinations were analyzed in lanes 1–4, 5–8, 9–12, 13–16, 17–20, and 21–24, respectively: SNY36-9A–pSN345, SNY110–pSN345, SNY80–pSN345, SNY36–9A–pSN346, SNY110–pSN346, and SNY80–pSN346. The positions of precursor (p) and processed mature (m) A-ALP are indicated.

Mentions: We first attempted to generate suppressor mutations in the cytosolic domain of A-ALP that would restore its retrieval in a vps35-101 strain. Vacuolar mislocalization of A-ALP caused by poor PVC retrieval results in processing of its COOH-terminal ALP propeptide by a vacuolar protease (Nothwehr et al. 1993). Upon processing, A-ALP becomes enzymatically active thus vacuolar processing can be detected by both an increase in gel mobility and by an increase in activity. Using PCR mutagenesis, a plasmid construct encoding A-ALP was randomly mutagenized in the region encoding the cytosolic domain. Other regions of the plasmid were unmutagenized. A library of vps35-101 yeast clones carrying the mutagenized plasmids was initially screened using an ALP activity assay (Chapman and Munro 1994; Nothwehr et al. 1996). Plasmids rescued from clones exhibiting reduced ALP activity and a slower rate of A-ALP processing were transformed back into the vps35-101 strain and into a vps35Δ strain to assess whether suppression was linked to the plasmid and was allele specific. Three plasmids were obtained that exhibited much slower processing in the vps35-101 strain but did not suppress the vps35Δ allele. In all three cases, the suppression phenotype was due to mutation of a single base pair as determined by DNA sequencing and restriction fragment swapping experiments. Interestingly, two of the mutations were at codon 88 (N88I and N88K) and the third was nearby at codon 92 (N92I) (Fig. 2 A).


Sorting of yeast membrane proteins into an endosome-to-Golgi pathway involves direct interaction of their cytosolic domains with Vps35p.

Nothwehr SF, Ha SA, Bruinsma P - J. Cell Biol. (2000)

(A) The positions of three independent mutations in the A-ALP cytoplasmic domain that specifically suppress the vps35-101 allele are shown. A schematic diagram of A-ALP is shown with the lumenal, transmembrane (TMD), and cytosolic domains indicated. The cytosolic domain residues previously shown to comprise a PVC-to-TGN retrieval signal (Nothwehr et al. 1993) are labeled with asterisks and the suppressor mutations (N88I, N88K, and N92I) are indicated with arrows. (B) The N88I mutation in the cytosolic domain of A-ALP reduces its rate of vacuolar processing in a vps35-101 strain. Yeast strains were pulse labeled with [35S]methionine/cysteine for 10 min and chased for the times indicated in minutes above the upper panel. The cells were lysed and wild-type or mutant A-ALP was immunoprecipitated and analyzed by SDS-PAGE and fluorography. The following strain–plasmid combinations were analyzed in lanes 1–4, 5–8, 9–12, 13–16, 17–20, and 21–24, respectively: SNY36-9A–pSN345, SNY110–pSN345, SNY80–pSN345, SNY36–9A–pSN346, SNY110–pSN346, and SNY80–pSN346. The positions of precursor (p) and processed mature (m) A-ALP are indicated.
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Figure 2: (A) The positions of three independent mutations in the A-ALP cytoplasmic domain that specifically suppress the vps35-101 allele are shown. A schematic diagram of A-ALP is shown with the lumenal, transmembrane (TMD), and cytosolic domains indicated. The cytosolic domain residues previously shown to comprise a PVC-to-TGN retrieval signal (Nothwehr et al. 1993) are labeled with asterisks and the suppressor mutations (N88I, N88K, and N92I) are indicated with arrows. (B) The N88I mutation in the cytosolic domain of A-ALP reduces its rate of vacuolar processing in a vps35-101 strain. Yeast strains were pulse labeled with [35S]methionine/cysteine for 10 min and chased for the times indicated in minutes above the upper panel. The cells were lysed and wild-type or mutant A-ALP was immunoprecipitated and analyzed by SDS-PAGE and fluorography. The following strain–plasmid combinations were analyzed in lanes 1–4, 5–8, 9–12, 13–16, 17–20, and 21–24, respectively: SNY36-9A–pSN345, SNY110–pSN345, SNY80–pSN345, SNY36–9A–pSN346, SNY110–pSN346, and SNY80–pSN346. The positions of precursor (p) and processed mature (m) A-ALP are indicated.
Mentions: We first attempted to generate suppressor mutations in the cytosolic domain of A-ALP that would restore its retrieval in a vps35-101 strain. Vacuolar mislocalization of A-ALP caused by poor PVC retrieval results in processing of its COOH-terminal ALP propeptide by a vacuolar protease (Nothwehr et al. 1993). Upon processing, A-ALP becomes enzymatically active thus vacuolar processing can be detected by both an increase in gel mobility and by an increase in activity. Using PCR mutagenesis, a plasmid construct encoding A-ALP was randomly mutagenized in the region encoding the cytosolic domain. Other regions of the plasmid were unmutagenized. A library of vps35-101 yeast clones carrying the mutagenized plasmids was initially screened using an ALP activity assay (Chapman and Munro 1994; Nothwehr et al. 1996). Plasmids rescued from clones exhibiting reduced ALP activity and a slower rate of A-ALP processing were transformed back into the vps35-101 strain and into a vps35Δ strain to assess whether suppression was linked to the plasmid and was allele specific. Three plasmids were obtained that exhibited much slower processing in the vps35-101 strain but did not suppress the vps35Δ allele. In all three cases, the suppression phenotype was due to mutation of a single base pair as determined by DNA sequencing and restriction fragment swapping experiments. Interestingly, two of the mutations were at codon 88 (N88I and N88K) and the third was nearby at codon 92 (N92I) (Fig. 2 A).

Bottom Line: By genetic and biochemical means we now show that Vps35p directly associates with the cytosolic domains of cargo proteins.Furthermore, mutations in the cytosolic domains of A-ALP and another cargo protein, Vps10p, were identified that suppressed cargo-specific mutations in Vps35p but did not suppress the retrieval defects of a vps35 mutation.Suppression was shown to be due to an improvement in protein sorting at the prevacuolar compartment.

View Article: PubMed Central - PubMed

Affiliation: Division of Biological Sciences, University of Missouri, Columbia, Missouri 65211, USA. nothwehrs@missouri.edu

ABSTRACT
Resident late-Golgi membrane proteins in Saccharomyces cerevisiae are selectively retrieved from a prevacuolar-endosomal compartment, a process dependent on aromatic amino acid-based sorting determinants on their cytosolic domains. The formation of retrograde vesicles from the prevacuolar compartment and the selective recruitment of vesicular cargo are thought to be mediated by a peripheral membrane retromer protein complex. We previously described mutations in one of the retromer subunit proteins, Vps35p, which caused cargo-specific defects in retrieval. By genetic and biochemical means we now show that Vps35p directly associates with the cytosolic domains of cargo proteins. Chemical cross-linking, followed by coimmunoprecipitation, demonstrated that Vps35p interacts with the cytosolic domain of A-ALP, a model late-Golgi membrane protein, in a retrieval signal-dependent manner. Furthermore, mutations in the cytosolic domains of A-ALP and another cargo protein, Vps10p, were identified that suppressed cargo-specific mutations in Vps35p but did not suppress the retrieval defects of a vps35 mutation. Suppression was shown to be due to an improvement in protein sorting at the prevacuolar compartment. These data strongly support a model in which Vps35p acts as a "receptor" protein for recognition of the retrieval signal domains of cargo proteins during their recruitment into retrograde vesicles.

Show MeSH
Related in: MedlinePlus