Despite this ambiguity, the Ca²⁺ concentration in the endoplasmic reticulum (ER) has been implicated in retention of resident luminal proteins (Booth and Koch, 1989 ), in export of secretory proteins (for review, see Sambrook, 1990 ), in protein folding and degradation (reviewed in Lodish and Kong, 1990 ; Wileman et al., 1991 ; Gething and Sambrook, 1992 ), and in the association of the ER chaperone BiP with misfolded proteins (Suzuki et al., 1991 ). Recent studies have demonstrated that some misfolded luminal proteins are exported from the ER into the cytosol for ubiquitination and subsequent degradation by the proteasome (for review, see Brodsky and McCracken, 1997 ; Sommer and Wolf, 1997 ). Yeast CpY*, a mutant form of carboxypeptidase Y (CpY), is subjected to this degradative route, and the ubiquitin-conjugating enzymes (Ubc6, Ubc7) have been identified (Hiller et al., 1996 ). In contrast, other non-native luminal proteins appear to exit the yeast ER via the secretory pathway and interact with Vps10, a transmembrane receptor normally engaged in sorting of soluble hydrolases, including CpY, from the Golgi to the vacuole (Marcusson et al., 1994 ; Cooper and Stevens, 1996 ). Apparently, Vps10 exerts a dual role in intracellular targeting, i.e., it participates in the sorting of specific vacuolar enzymes and in a more general salvage pathway guiding non-native luminal proteins to the vacuole (Hong et al., 1996 ). It is not known whether Ca²⁺ ions in the ER affect export of substrates for degradation by the proteasome. Similarly, it is unclear how intralumenal Ca²⁺ influences the Vps10-mediated salvage pathway.

Accumulation of unfolded proteins in the ER, experimentally induced by the addition of tunicamycin to block glycosylation or by treatment with reducing agents such as DTT, activates a universal signal transduction cascade that allows eukaryotic cells to alter the conditions in the ER (Lee, 1987 ; Kozutsumi et al., 1988 ). This unfolded protein response (UPR), best understood in the yeast Saccharomyes cerevisiae (for review, see Shamu et al., 1994 ; Nunnari and Walter, 1996 ; Shamu, 1997 ), coordinates the transcription of genes encoding ER-resident chaperones with the synthesis of glycerophospholipids destined for the ER membrane (Cox et al., 1997 ). Inactivation of UPR components, like Ire1 (a transmembrane kinase of the ER and/or inner nuclear membrane) or Hac1 (a transcription factor binding to UPR-regulated promoters), leads to a complete block of the UPR. Such yeast mutants are sensitive to DTT and tunicamycin and fail to grow on media lacking inositol, a precursor in the synthesis of phosphatidylinositol.

Within the Golgi complex, high Ca²⁺ concentrations are found in all cisternae along the cis–trans axis (Pezzati et al., 1997 ). The available evidence for a role of Ca²⁺ in important Golgi functions, including luminal and membrane protein traffic (Carnell and Moore, 1994 ), cargo condensation, and precursor processing (Chanat and Huttner, 1991 ; Fuller et al., 1989 ; Oda, 1992 ), suggests that Golgi Ca²⁺ transport and storage are specifically regulated, independently from the control of ER Ca²⁺. As inferred from the in vitro requirement for Mn²⁺ by numerous enzymes involved in oligosaccharide addition in animal cells and yeast, the Golgi complex hosts reactions that depend on Mn²⁺ (Sharma et al., 1974 ; Nakajima and Ballou, 1975 ; Parodi, 1979 ; Sugiura et al., 1982 ; Elhammer and Kornfeld, 1986 ; Haselbeck and Schekman, 1986 ). Depletion of manganese in vivo inhibits O-linked and N-linked protein glycosylation in mammalian cells (Kaufman et al., 1994 ). Similar results were obtained with thapsigargin, a potent inhibitor of the SERCA-type Ca²⁺-adenosine triphosphatases present in sarco/endoplasmic reticulum membranes, suggesting a role of these ion pumps in maintaining intralumenal Ca²⁺ and Mn²⁺ concentrations (Kaufman et al., 1994 ).

In yeast, the only Ca²⁺ pump known to reside in the early secretory pathway (ER, Golgi) is Pmr1 (Rudolph et al., 1989 ; Antebi and Fink, 1992 ; Cunningham and Fink, 1994 , 1996 ; Sorin et al., 1997 ). In subcellular fractionation and immunofluorescence microscopy, Pmr1 almost entirely coincides (> 98%) with the steady-state pool of Emp47, a lectine-like transmembrane protein of the medial-Golgi, which recycles, in contrast to Pmr1, between the ER and the Golgi (Schröder et al., 1995 ). These findings, consistent with previous work demonstrating a Golgi-like distribution for Pmr1 (Antebi and Fink, 1992 ), imply that only minute amounts of Pmr1 reside in the ER membrane. Since no other candidate gene that could encode an ATP-driven Ca²⁺ pump of the ER, is present in the genome of Saccharomyces cerevisiae, the yeast ER appears to lack a spezialized Ca²⁺ pump (Sorin et al., 1997 ). This raises interesting questions on how yeast cells achieve the appropriate distribution of Ca²⁺ and Mn²⁺ ions within organelles of the early secretory pathway, which is thought to critically depend on the adequate presence of both ions.

The function of Pmr1 as an ATP-driven Ca²⁺ pump was recently demonstrated by assaying Ca²⁺ uptake into purified Golgi-derived vesicles (Sorin et al., 1997 ). Genetically, the role of Pmr1 in Ca²⁺ transport is manifest in pmr1 mutants in several Ca²⁺-related phenotypes, one of which is a partial defect in CpY sorting. In media with approximately 200 μM Ca²⁺, pmr1 cells mis-sort and secrete a small fraction (< 7%) of the Golgi form of CpY, normally routed to the vacuole in wild-type cells. Addition of Ca²⁺ (10 mM) fully restores CpY sorting (Antebi and Fink, 1992 ). In addition, the free cytosolic Ca²⁺ in pmr1 cells is known to be elevated relative to wild type, and cytosolic Ca²⁺ increases even further upon addition of extracellular Ca²⁺ (Halachmi and Eilam, 1996 ). Therefore, the reversibility of the CpY-sorting defect in pmr1 cells by external Ca²⁺ suggests that a low intralumenal Ca²⁺ content within some secretory organelle(s), rather than an elevated cytosolic Ca²⁺ concentration, is causing mis-sorting of CpY. This view is now corroborated by our finding that severe Ca²⁺ depletion in wild-type cells induces the same effect on CpY sorting as a pmr1 mutation, i.e., partial secretion of CpY. These data also provide a clear demonstration that chelators can effectively induce cation depletion in secretory organelles of growing yeast cells.

The aberrant secretion of CpY from wild-type cells is not caused by the Mn²⁺ ions present in the Ca²⁺-depleted medium to support growth, since chelator-free medium with a similar concentration of Mn²⁺ allows for accurate sorting. In addition, the growth rates in Ca²⁺- or Mn²⁺-depleted media are very similar, suggesting that either cation alone does not drastically alter bulk flow through the secretory pathway. Based on the ER-associated changes we observed in pmr1 cells (see Figure ​Figure5),5), it seems likely that Ca²⁺ depletion leads to an increased production or reduced turnover of malfolded proteins, which might then utilize the Vps10-mediated salvage pathway to the vacuole and could thereby compete with CpY for binding to the Vps10 receptor. Likewise, the biogenesis or function of Vps10 might be compromised under low intralumenal Ca²⁺ conditions. Alternatively, luminal Ca²⁺ could directly promote specific sorting steps, such as partitioning of receptor–cargo complexes into a budding vesicle or binding of CpY to Vps10, but the Ca²⁺ dependence of these reactions has not been explored in vitro (for binding of CpY to Vps10, see Cooper and Stevens, 1996 ).

A role of Pmr1 in Mn²⁺ transport was first suggested based on changes in intracellular Mn²⁺ distribution observed in pmr1 cells, which appear to possess an increased cytosolic Mn²⁺ level (Lapinskas et al., 1995 ). The present study extends this observation by showing that pmr1 cells display Mn²⁺-related defects in glycosylation reactions that take place in the lumen of the Golgi. We have shown that chitinase, a solely O-glycosylated secretory protein, is produced by pmr1 cells in an aberrant form migrating during SDS-PAGE significantly faster than chitinase isolated from a wild-type strain. A similar behavior was previously reported for N-glycosylated invertase, which in pmr1 cells lacks the complex carbohydrate chains normally added in the Golgi (Antebi and Fink, 1992 ). As we have shown, addition of Mn²⁺ stimulates pmr1 cells to produce high-molecular mass forms of chitinase and invertase. Since the mannosyltransferase activities responsible for carbohydrate addition onto N- and O-glycosylated precursors in the yeast Golgi require Mn²⁺ in vitro (Sharma et al., 1974 ; Nakajima and Ballou, 1975 ; Parodi, 1979 ; Haselbeck and Schekman, 1986 ), we assume that the altered molecular mass in the different forms of chitinase and invertase reflects changes in carbohydrate content, although this has only been demonstrated for invertase produced by pmr1 cells (Antebi and Fink, 1992 ).

Glycosylation reactions in the Golgi complex of mammalian cells display similar Mn²⁺ requirements (Sugiura et al., 1982 ; Elhammer and Kornfeld, 1986 ). In one study, defects in O- and N-linked glycosylation were generated in vivo by A23187-induced cation depletion and shown to be partially reversible by the addition of Mn²⁺, but not Ca²⁺. Interestingly, Mn²⁺ addition in the presence of A23187 produced two distinct populations of secreted molecules (macrophage colony-stimulating factor): one form carried fully restored complex N-linked and O-linked carbohydrates; the other one lacked O-linked oligosaccharides, but displayed high-mannose N-linked carbohydrates (Kaufman et al., 1994 ). Our results with chitinase produced upon Mn²⁺ addition are very similar: pmr1 cells also secrete two populations of chitinase under these conditions. This “all or none” behavior in glycosylation could reflect secretion of chitinase from different secretory compartments or point to some other defect within the secretory pathway, presumably caused by a partial Ca²⁺ depletion in the absence of Pmr1. The appearance of a single, intermediate form of chitinase upon addition of Ca²⁺ to pmr1 cells favors this explanation. Likewise, Mn²⁺ addition does not restore full glycosylation onto invertase in pmr1 cells, and the sole addition of Ca²⁺ appears to have a slight stimulatory effect. Thus, the faithful addition of carbohydrates onto O- and N-glycosylated proteins seems to require an intricate balance of intralumenal Ca²⁺ and Mn²⁺ levels within the secretory pathway. We have also noticed that the two pmr1 strains, derived from a common his3 leu2 parent strain by transformation, produce the two chitinase forms in different amounts (see Figure ​Figure2B).2B). The particulary strong response to Mn²⁺ observed in the pmr1::LEU2 strain could be due to the his3 mutation, which is complemented in the pmr1::HIS3 strain. Histidine is a fairly good chelator of Mn²⁺ with a dissociation constant of 20 nM (see Hughes and Poole, 1991 , and references therein), and is stored in high amounts by yeast vacuoles (Kitamoto et al., 1988 ), which also accumulate divalent cations, including Ca²⁺, Mg²⁺, Mn²⁺, and other heavy metals (Okorokov et al., 1978 ; Ohsumi and Anraku, 1983 ; Bode et al., 1995 ). The ability to use the full capacity of the endogenous biosynthetic pathway to supply histidine for sequestration of Mn²⁺ into the vacuole might, to some extent, attenuate the high Mn²⁺ conditions in pmr1::HIS3 cells, and thereby indirectly affect chitinase glycosylation.

Two sets of data, one of which relates to the failure of pmr1 cells to effectively degrade CpY*, indicate that Pmr1-mediated ion transport affects intralumenal processes hosted in a secretory compartment outside the Golgi, presumably within the ER. The observed stabilization of CpY* appears to reflect an intralumenal defect, since the same pmr1 strain is able to degrade cytosolic Deg1-β-galactosidase. Deg1-β-galactosidase is, like CpY* (Hiller et al., 1996 ), an Ubc6-/Ubc7-dependent substrate of the proteasome due to the presence of the Deg1 domain, which is responsible for ubiquitination of Matα2 by the ubiquitin-conjugating enzymes Ubc6 and Ubc7 (Chen et al., 1993 ). Overexpression of the luminal domain of Vps10 (Vps10–1385), which we demonstrated to suppress EGTA hypersensitivity in pmr1 strains, does not affect the high steady-state level of CpY* in pmr1 cells, indicating that CpY* accumulation is not indirectly caused by a potentially Ca²⁺-controlled redistribution of malfolded proteins between Vps10-dependent and -independent degradative pathways (Plemper, Strayle, Wolf, and Rudolph, unpublished data). In contrast to this, expression of the SERCA1a Ca²⁺ pump, which in PMR1 wild-type cells accumulates in proliferating ER membranes (Catty, unpublished data), reduces the steady-state level of CpY* in pmr1 cells (Plemper, Strayle, Wolf, and Rudolph, unpublished data). These findings suggest that a sufficient level of ER Ca²⁺ is necessary to accomplish export of CpY* into the cytosol for degradation by the proteasome. A requirement for Kar2, the yeast BiP homolog, in CpY* export has been demonstrated (Plemper et al., 1997 ). Thus, the observed effects of Pmr1 on CpY* degradation could result from cation-dependent functions of the Kar2 chaperone. Remarkably, mammalian BiP was shown to undergo autophosphorylation, which in vitro is stimulated by Ca²⁺ and inhibited by Mn²⁺, and the resulting BiP isoforms display altered properties in protein binding and oligomerization in vivo (Hendershot et al., 1988 ; Leustek et al., 1991 ; Carlino et al., 1992 ). Similar, cation-dependent changes in Kar2 phosphorylation could reduce the level of Kar2 available for CpY* export and presumably affect other Kar2-mediated reactions, including protein folding.

The second set of data pointing to an ER-related function of Pmr1 concerns the EGTA hypersensitivity of pmr1 cells. EGTA and BAPTA both lower the divalent cation content of secretory organelles in animal cells, and the partial mis-sorting of CpY we observed here with wild-type cells provides direct evidence for a similar effect of chelators on yeast cells. The strong suppression of EGTA hypersensitivity by Vps10–1385, an entirely luminal polypeptide secreted under these conditions, further confirms the inferred intralumenal nature of the EGTA-induced growth inhibition in pmr1 cells. Based on the function of Vps10 in salvage of non-native luminal proteins to the vacuole (Hong et al., 1996 ), we hypothesize that EGTA-induced cation depletion in some secretory organelle(s) of pmr1 cells might generate malfolded proteins, which above a certain threshold could cause growth inhibition. As shown here, accumulation of CpY* is already occurring during growth of pmr1 cells in a normal ionic milieu, indicating that the ER of pmr1 cells is particularly sensitive to cation depletion. Thus, we suspect that the EGTA-induced growth inhibition originates in this compartment. It is not clear, however, whether reduced export of misfolded proteins from the ER to the cytosol, a prerequisite for degradation of CpY* and perhaps other malfolded ER proteins, is responsible for the growth inhibtion by EGTA. Alternatively, a low cation content in the ER could compromise protein folding to an extent exceeding the capacity of the Vps10 salvage pathway, which might also be affected under these conditions. Nevertheless, expression of either Ca²⁺ pump, yeast Pmc1, or heterologous SERCA1a restores growth of pmr1 cells in the presence of EGTA, suggesting that the mechanisms underlying EGTA hypersensitivity are Ca²⁺-dependent.

Interestingly, the loss of Pmr1 leads to phenotypes also observed in yeast mutants with a blocked UPR, which coordinates synthesis of ER-resident chaperones and ER membrane biogenesis under a variety of ER stress conditions (Cox et al., 1997 ; see Shamu, 1997 and references therein). As we have shown, induction of Kar2 after tunicamycin challenge proceeds unperturbed for at least 3 h in pmr1 cells, suggesting that the regulatory branch in the UPR to increase chaperone synthesis is functional. It needs to be tested whether pmr1 cells are unable to sustain prolonged induction of chaperone synthesis or fail to induce INO1, the gene encoding inositol-1-phosphate synthase required for synthesis of phosphatidylinositol. It is also possible that an altered cation distribution in pmr1 cells indirectly affects phospholipid biosynthetic enzymes requiring Mg²⁺, Mn²⁺, or, as in one case, Ca²⁺ (see Paltauf et al., 1992 and references therein). Based on our study, which emphasizes the importance of Pmr1-mediated ion transport for early secretory organelles, including the ER, we also suspect that the Pmr1 ion pump might be directly required during UPR to uphold a favorable ionic milieu within an expanding ER.