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. 1997 Aug 11;138(3):531-45.
doi: 10.1083/jcb.138.3.531.

The membrane protein alkaline phosphatase is delivered to the vacuole by a route that is distinct from the VPS-dependent pathway

R C Piper  1 , N J Bryant, T H Stevens
Affiliations

The membrane protein alkaline phosphatase is delivered to the vacuole by a route that is distinct from the VPS-dependent pathway

R C Piper et al. J Cell Biol. .

Abstract

Membrane trafficking intermediates involved in the transport of proteins between the TGN and the lysosome-like vacuole in the yeast Saccharomyces cerevisiae can be accumulated in various vps mutants. Loss of function of Vps45p, an Sec1p-like protein required for the fusion of Golgi-derived transport vesicles with the prevacuolar/endosomal compartment (PVC), results in an accumulation of post-Golgi transport vesicles. Similarly, loss of VPS27 function results in an accumulation of the PVC since this gene is required for traffic out of this compartment. The vacuolar ATPase subunit Vph1p transits to the vacuole in the Golgi-derived transport vesicles, as defined by mutations in VPS45, and through the PVC, as defined by mutations in VPS27. In this study we demonstrate that, whereas VPS45 and VPS27 are required for the vacuolar delivery of several membrane proteins, the vacuolar membrane protein alkaline phosphatase (ALP) reaches its final destination without the function of these two genes. Using a series of ALP derivatives, we find that the information to specify the entry of ALP into this alternative pathway to the vacuole is contained within its cytosolic tail, in the 13 residues adjacent to the transmembrane domain, and loss of this sorting determinant results in a protein that follows the VPS-dependent pathway to the vacuole. Using a combination of immunofluorescence localization and pulse/chase immunoprecipitation analysis, we demonstrate that, in addition to ALP, the vacuolar syntaxin Vam3p also follows this VPS45/27-independent pathway to the vacuole. In addition, the function of Vam3p is required for membrane traffic along the VPS-independent pathway.

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Figures

Figure 1
Figure 1
Differential localization of ALP and other vacuolar proteins. (A) Double label immunofluorescence of Vph1p (upper panels) and ALP (middle panels) is shown for wild-type cells (SF838-9D), vps45Δ cells (RPY12), and vps27Δ cells (HYY1). The depressions visible in the Nomarski image (lower panels) identify the yeast vacuole. (B) Immunoprecipitation of newly synthesized ALP and the soluble vacuolar hydrolase CPY in wild-type cells (RPY10), vps45Δ cells (RPY11), and vps27Δ cells (AACY5). Newly synthesized proteins were labeled for 10 min at 30°C with the addition of 35S-Express. Excess unlabeled methio-nine and cysteine were then added for the indicated times. After centrifugation, internal (pellet) and external (supernatant) fractions were then prepared. CPY was immunoprecipitated from intracellular and extracellular fractions and ALP was immunoprecipitated from intracellular fractions. The PEP4-dependent cleavage products of ALP are indicated (*).
Figure 2
Figure 2
Differential targeting of ALP and Vph1p in temperature-sensitive vps mutants. The targeting of newly synthesized Vph1p and ALP was monitored in vps45-ts cells (RPY94; top panels), vps27-ts cells (RPY90; middle panels), and wild-type cells (RPY111; bottom panels). The production of both ALP and Vph1p in RPY94, RPY90, and RPY111 cells was under the control of the galactose-inducible GAL1 promoter. Cells were grown overnight at 22°C in the absence of galactose. Galactose was then added, and cells were incubated for 45 min at 22°C or immediately shifted to 37°C as indicated. Cycloheximide was then added (100 μg/ml) for an additional 10 min before fixation and double labeled for Vph1p and ALP using indirect immunofluorescence. Vph1p was labeled with rabbit anti-Vph1p antibodies and Texas red–conjugated secondary antibody (left panels); ALP was labeled with anti-ALP 1D3 mAb with biotinylated secondary and FITC-conjugated streptavidin. Shown as a control are wild-type cells (RPY111) incubated at 37°C in the presence or absence of galactose (lower panels).
Figure 3
Figure 3
Trafficking of ALP and Vps10p-Δ10* in temperature-sensitive vps mutants. (A) The rate of PEP4-dependent processing of Vps10p-10* (left panels) and ALP (right panels) was measured in wild-type cells (RPY96) and vps45Δ cells (RPY99) by pulse/chase analysis. Cells were labeled with 35S-Express for 10 min at 30°C and chased for the indicated times. Cell lysates were divided and subjected to immunoprecipitation with anti-ALP and anti-Vps10p antibodies. Both the full-length Vps10p and the truncated Vps10p-Δ10* were present. The PEP4-dependent cleavage products of Vps10p-Δ10* and ALP are indicated (*). (B) The rate of PEP4-dependent cleavage of Vps10p-Δ10* and ALP was measured in vps45-ts cells (RPY102). Cells were grown overnight at 22°C and then either maintained at 22°C or shifted to 37°C 10 min before labeling with 35S-Express for 10 min. The chase times used are indicated. ALP and Vps10p-Δ10* were immunoprecipitated as described above. The rate of PEP4-dependent cleavage of Vps10p-Δ10* and ALP was measured in vps27-ts cells (RPY103) and wild-type cells (RPY110) each carrying the GAL1-PEP4 plasmid, pRCP39. Cells were grown in galactose-containing media for 24 h at 22°C and then shifted to glucose-containing media for 24 h before the pulse/chase immunoprecipitation scheme used for vps45-ts cells (RPY102) above. Cells were either maintained at 22°C or shifted to 37°C 10 min before labeling.
Figure 4
Figure 4
ALP does not travel to the cell surface in vps45Δ cells. The rate of PEP4-dependent cleavage of ALP was measured in wild-type cells (RPY96), sec1-ts cells (RPY109), sec1-ts vps45Δ cells (YM), and sec1-ts vps1Δ cells (SNY31). Cells were grown overnight at 22°C and either maintained at 22°C or shifted to 37°C for 15 min before labeling for 10 min with 35S- Express. Label was chased for the indicated times. After centrifugation, internal (pellet) and external (supernatant) fractions were prepared, divided, and subjected to immunoprecipitation with anti-CPY and anti-ALP antibodies.
Figure 5
Figure 5
The vacuolar syntaxin Vam3p is required for the alternative bypass pathway. (A) Localization of Vam3p in vps27Δ cells. Vam3p was labeled with rabbit anti-Vam3p antibodies, biotinylated secondary and FITC-conjugated streptavidin (middle panels). As a marker of the class E prevacuolar compartment, the 60-kD subunit of the vacuolar ATPase, Vma2p, was localized with the mAb 13D11B2 and Texas red–conjugated secondary antibody (left panels). (B) The rate of PEP4-dependent processing of Vps10p-10* (left panels) and ALP (right panels) was measured in vam3Δ cells (RPY95). Cells were labeled with 35S-Express for 10 min at 30°C and chased for the indicated times. Cell lysates were divided and subjected to immunoprecipitation with anti-ALP and anti-Vps10p antibodies. (C) The rate of PEP4-dependent processing of Vps10p-10* (left panels) and ALP (right panels) was measured in wild-type cells (RPY96) carrying the dominant-interfering allele VAM3-TMΔ (pRCP161) under the inducible control of the GAL1 promoter. pRCP161 expresses the cytosolic domain of Vam3p in response to the addition of galactose. Cells were grown overnight at 30°C in raffinose. Galactose was then added for the indicated times (no addition, 15 min, or 30 min) before labeling cells with 35S-Express for 10 min at 30°C. Label was chased for the indicated times and cell lysates were divided and subjected to immunoprecipitation with anti-ALP and anti-Vps10p antibodies.
Figure 6
Figure 6
The cytosolic domain of ALP is necessary for entry into the alternative bypass pathway. (A) Schematic description of the ALP mutant proteins analyzed. Truncations in the NH2-terminal cytosolic tail of ALP were made by deleting the first 21 or 31 codons of the ALP open reading frame to generate pNB3 (Δ2–21 ALP) and pNB4 (Δ2–31 ALP). The (F/A) A-ALP protein consists of the luminal and transmembrane domains of ALP fused to the cytosolic tail of Ste13p in which the Golgi localization motif FXFXD has been ablated by the mutation AXAXD. (B) The distribution of the mutant ALP proteins (wild-type ALP, Δ2–21, Δ2–31, and [F/A] A-ALP) was assessed in vps27Δ pho8 cells (CKRY2-8A) transformed with CEN-URA3 plasmids, pSN92 (ALP), pNB3 (Δ2–21-ALP), pNB4 (Δ2–31-ALP), and pSN100 ([F/A] A-ALP) using immunofluorescence. Transformants were fixed and labeled with anti-ALP antibodies together with biotinylated secondary antibodies and FITC-conjugated streptavidin. (C) The PEP4-dependent cleavage of the mutant ALP proteins (wild-type ALP, Δ2–21, Δ2–31, and [F/A] A-ALP) was assessed in vps45Δ pho8Δ cells (NBY68) using pulse/chase analysis. Transformants were labeled for 10 min with 35S-Express and chased for the indicated times, after which time lysates were subjected to immunoprecipitation with anti-ALP antibodies. (D) Schematic representation of the cytosolic tail of ALP. (Arrowheads) Second residue (after methionine) of the deletion constructs.
Figure 7
Figure 7
The cytosolic domain of ALP can confer sorting into the alternative bypass pathway. The schematic (right) shows the wild-type DPAP B protein (DPAP B), a chimeric protein comprised of the cytosolic domain of ALP fused to the transmembrane and luminal domains of DPAP B (ALP/DPAP B), and an ALP/DPAP B chimeric protein with a deletion of amino acids 2–11 of the ALP cytosolic domain. All constructs (pCJR6, pAIN1, and pAIN2, respectively) were under the control of the GAL1 promoter. The targeting of these proteins was analyzed by immunofluorescence (left) in vps27 dap2Δ cells (RPY107) transformed with pCJR6 or pAIN1. Transformants were grown overnight in raffinose, shifted to 2% galactose for 1 h, fixed, and then processed for immunofluorescence labeling using anti–DPAP B antibodies, biotinylated secondary antibody, and FITC-conjugated streptavidin.
Figure 8
Figure 8
Model for the VPS-dependent pathway and the alternative bypass pathway to the vacuole. Proteins such as the CPY receptor and Vph1p enter vesicles in the late Golgi (TGN) of yeast that fuse with a PVC. This fusion event is controlled by Vps45p. The CPY receptor then recycles back to the Golgi while proteins such as Vph1p and CPY are delivered to the vacuole. These latter steps are controlled by Vps27p. ALP bypasses these intermediates and neither enters the Vps45p-controlled Golgi-derived transport vesicles nor transits through the PVC. ALP may enter a specialized transport vesicle that fuses directly with the vacuole or may be transported via a yet unidentified intermediate compartment (?).
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References

    1. Aalto MK, Keranen S, Ronne H. A family of proteins involved in intracellular transport. Cell. 1992;68:181–182. - PubMed
    1. Bankaitis VA, Johnson LM, Emr SD. Isolation of yeast mutants defective in protein targeting to the vacuole. Proc Natl Acad Sci USA. 1986;83:9075–9079. - PMC - PubMed
    1. Banta LM, Vida TA, Herman PK, Emr SD. Characterization of yeast Vps33p, a protein required for vacuolar protein sorting and vacuole biogenesis. Mol Cell Biol. 1990;10:4638–4649. - PMC - PubMed
    1. Bauerfeind R, Ohashi M, Huttner WB. Biogenesis of secretory granules and synaptic vesicles. Facts and hypotheses. Ann NY Acad Sci. 1994;733:233–244. - PubMed
    1. Becherer KA, Rieder SE, Emr SD, Jones EW. Novel syntaxin homolog, Pep12p, required for the sorting of lumenal hydrolases to the lysosome-like vacuole in yeast. Mol Biol Cell. 1996;7:579–594. - PMC - PubMed

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