The medial-Golgi ion pump Pmr1 supplies the yeast secretory pathway with Ca2+ and Mn2+ required for glycosylation, sorting, and endoplasmic reticulum-associated protein degradation

Abstract

The yeast Ca2+ adenosine triphosphatase Pmr1, located in medial-Golgi, has been implicated in intracellular transport of Ca2+ and Mn2+ ions. We show here that addition of Mn2+ greatly alleviates defects of pmr1 mutants in N-linked and O-linked protein glycosylation. In contrast, accurate sorting of carboxypeptidase Y (CpY) to the vacuole requires a sufficient supply of intralumenal Ca2+. Most remarkably, pmr1 mutants are also unable to degrade CpY*, a misfolded soluble endoplasmic reticulum protein, and display phenotypes similar to mutants defective in the stress response to malfolded endoplasmic reticulum proteins. Growth inhibition of pmr1 mutants on Ca2+-deficient media is overcome by expression of other Ca2+ pumps, including a SERCA-type Ca2+ adenosine triphosphatase from rabbit, or by Vps10, a sorting receptor guiding non-native luminal proteins to the vacuole. Our analysis corroborates the dual function of Pmr1 in Ca2+ and Mn2+ transport and establishes a novel role of this secretory pathway pump in endoplasmic reticulum-associated processes.

Figure 1

Growth and vacuolar sorting of S. cerevisiae in ion-depleted media. (A) Growth in Ca²⁺- or Mn²⁺-depleted media. Wild-type strain YR98 was inoculated into media containing BAPTA to buffer the free ion concentrations of Ca²⁺ and Mn²⁺ as described. Media: −Mn²⁺ medium (1.4 × 10⁻⁶ M Ca²⁺, 1.6 × 10⁻¹³ M Mn²⁺), −Ca²⁺ medium (8.4 × 10⁻¹⁰ M Ca²⁺, 2.0 × 10⁻⁸ M Mn²⁺), −Mn²⁺, −Ca²⁺ medium (8.4 × 10⁻¹⁰ M Ca²⁺, 1.8 × 10⁻¹¹ M Mn²⁺). For the BAPTA-free control medium (−BAPTA), total ion concentrations (5.7 × 10⁻⁶ M Ca²⁺; 7.3 × 10⁻⁸ M Mn²⁺) were determined. (B) Depletion of Ca²⁺, but not Mn²⁺, induces partial secretion of CpY in wild type. PMR1 cells (YR98), pregrown in the defined media given in panel A, were converted to spheroblasts and labeled with ³⁵S-methionine maintaining the defined free ion concentrations. After 20 min and 40 min, supernatants were analyzed for the presence of total CpY by immunoprecipitation, SDS-PAGE, and autoradiography. All samples showed a single band migrating with the molecular mass (69 kDa) of the Golgi form, p2 CpY. Each lane corresponds to 3.5 OD of cells.

Figure 2

Mn²⁺ ions effectively stimulate glycosylation in pmr1 mutants. (A) N-glycosylated invertase secreted by wild-type (PMR1) and pmr1 mutant cells in unsupplemented medium (−), and after stimulation with Ca²⁺ or Mn²⁺ ions. Strains were pregrown in YPD (5% glucose), transferred to YPD (5% glucose, 1 h), supplemented with Ca²⁺ or Mn²⁺, and induced for invertase production in YPD (0.1% glucose, 1 h) supplemented with Ca²⁺ or Mn²⁺ as indicated. Control cells were grown and induced for invertase in unsupplemented media (−). Analysis of external invertase by native-gel electrophoresis and subsequent activity staining was as described (Rudolph et al., 1989). (B) O-glycosylated chitinase secreted by wild-type and pmr1 cells upon stimulation with Ca²⁺ or Mn²⁺. Cultures were grown overnight in YPD medium supplemented with Ca²⁺ or Mn²⁺ as indicated; control cells were grown in unsupplemented YPD medium (−). Secreted chitinase was affinity-purified using chitin powder (Guthrie and Fink, 1991) and analyzed by SDS-PAGE and Western blotting as described (Immervoll et al., 1995). Strains: YR98 (PMR1), YR122 (pmr1-Δ1::LEU2), YR123 (pmr1-Δ2::HIS3)

Figure 3

Expression of Ca²⁺ pumps restores growth of a pmr1 mutant on EGTA-containing media. (A) Expression of the vacuolar ion pump Pmc1 in pmr1 cells. Serial fivefold dilutions of saturated cultures were spotted onto complete medium lacking uracil to maintain selection for the plasmids. Addition (+) or omission (−) of 6.5 mM EGTA is indicated. Plates were photographed after 3 d incubation at 30°C. Strains (from left to right): YR439 (pmr1; 2μ-vector); YR657 (pmr1, 2μ-PMR1) and YGD57 (pmr1, 2μ-PMC1). (B) Expression of a sarcoplasmic Ca²⁺ pump, SERCA1a (rabbit). Conditions as in panel A; strains are (from left to right): YR441 (pmr1, 2μ-PMR1), YR439 (pmr1, 2μ-vector), YR663 (pmr1, CEN-SERCA1a), and YR664 (pmr1, 2μ-SERCA1a).

Figure 4

Suppression of EGTA hypersensitivity in pmr1 cells by the sorting receptor Vps10 is mediated through its luminal domain. (A) Suppression by wild-type Vps10. Serial fivefold dilutions of saturated cultures were spotted onto complete medium lacking uracil. Addition (+) or omission (−) of 6.5 mM EGTA is indicated. Plates were photographed after 3 d incubation at 30°C; strains are (left to right): YR439 (pmr1, 2μ-vector), YR657 (pmr1, 2μ-PMR1), and YR480 (pmr1, 2μ-VPS10). (B) Vps10-mediated suppression does not require Pmc1. Conditions are as in panel A. Strains (from left to right): YR469 (pmr1 pmc1 cnb1, 2μ-vector), YR472 (pmr1 pmc1 cnb1, 2μ-PMR1), and YR477 (pmr1 pmc1 cnb1, 2μ-VPS10). (C) The luminal domain of Vps10 is necessary and sufficient for suppression. Same conditions as in panel A; strains are (from left to right): YR547 (pmr1, 2μ-vector), YR551 (pmr1, 2μ-VPS10), YR550 (pmr1, 2μ-VPS10–1385), and YR549 (pmr1, 2μ-SUC::VPS10-ΔN). (D) Vps10 derivatives used in the analysis. The transmembrane topology of Vps10 and its derivatives is depicted; numbers indicate the Vps10 residues present in each derivative. (E) Vps10–1385 is secreted by pmr1 cells during growth in EGTA-containing medium. Cells were cultured in synthetic complete medium, converted to spheroblasts, and labeled (15 μCi/OD cells) with ³⁵S-methionine as described (Horazdovsky and Emr, 1993), but EGTA (3 mM) was present during these steps. At the times indicated, cells and media supernatants were analyzed for the presence of Vps10–1385 by immunoprecipitation, SDS-PAGE, and autoradiography. The band corresponding to Vps10–1385 in SDS-PAGE is marked (arrow). Strains: YR547 (pmr1, 2μ-vector) and YR550 (pmr1, 2μ-VPS10–1385).

Figure 5

Pmr1 is required for ER-associated degradation. (A) CpY* is stabilized in the pmr1 mutant. Pulse-chase analysis of CpY* was performed using the congenic strains W303–1C (wild type, ▪) and YRP023 (Δpmr1, ♦). Cells were grown at 30°C. At the indicated chase times, cells were lysed, and CpY* was immunoprecipitated. Antigenic material was separated by SDS-PAGE; the band corresponding to CpY* is indicated. (B) Quantification of the results shown in panel A using a Molecular Dynamics imaging system. (C) The Ubc6/Ubc7-dependent proteasome degradation system is functional in pmr1. β-Galactosidase activity was tested after alkaline lysis of transformants of W303–1C (PMR1, ▪) and YRP023 (Δpmr1, ♦) expressing the plasmid-encoded fusion protein Deg1-β-galactosidase (Chen et al., 1993).

Figure 6

Mutations in PMR1 affect the response to induced accumulation of malfolded proteins in the ER. (A) The pmr1 mutant is sensitive to DTT. PMR1 (YR98) and pmr1 (YR122) cells were streaked onto YPD media, supplemented with 0.2% DTT (+) or without addition of DTT (−). Plates were photographed after 3 d incubation at 30°C. (B) The pmr1 mutant displays inositol auxotrophy and hypersensitivity to tunicamycin. Serial fivefold dilutions of saturated cultures were spotted onto synthetic media (left panel) containing (+) or lacking (−) inositol and onto YPD media containing (+) or lacking (−) tunicamycin (0.3 μg/ml, right panel). Plates were photographed after 3 d incubation at 30°C. Strains: YR98 (PMR1) and YR122 (pmr1). (C) Induction of Kar2 after treatment with tunicamycin. Tunicamycin (1 μg/ml) was added to cells growing in YPD (OD₆₀₀ = 0.8). After 1 h, 2 h, and 3 h, cell extracts were prepared and analyzed for the presence of Kar2 by SDS-PAGE and Western blotting. Each lane corresponds to 20 μg of total protein. Strains: YR98 (PMR1) and YR122 (pmr1). (D) PhosphoImager quantitation of the results shown in panel C. For each strain, signals are expressed as ratio of relative induction; the values at t = 0 were arbitrarily set to 1. Strains: YR98 (PMR1) and YR122 (pmr1).

Figure 7

Model for the function of the secretory pathway pump Pmr1. Pmr1 pumps Ca²⁺ and Mn²⁺ into the secretory pathway, predominantly at the medial-Golgi (Pmr1, boldface). Both ions could either uniformly enter transport vesicles or be selectively recruited (or excluded). Such mechanisms could serve to enrich Ca²⁺ in the ER, or to reduce Mn²⁺, despite the use of a single pump to transport both ions. In the ER, which contains only a small amount of Pmr1 in the membrane (Pmr1, small font), Pmr1 activity is required to export malfolded proteins into the cytosol for degradation, but other processes are likely to be affected (folding, UPR). In the Golgi, Mn²⁺ is required for protein glycosylation, whereas Ca²⁺, directly or indirectly, sustains vacuolar sorting. Thus, Pmr1 could control the secretory pathway at several stages. Reduced Pmr1 activity could induce the secretion of nonnative proteins, otherwise retained for ER-associated degradation or salvaged by the vacuole; the first isolation of a pmr1 mutant in a screen for “supersecretion” of heterologous proteins from yeast supports this view (Smith et al., 1985). Abbreviations used: N, nucleus; ER, endoplasmic reticulum; V, vacuole; PM, plasma membrane.