Themedial-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 ofpmr1 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.

Functional expression of heterologous proteins in yeast: insights into Ca2+ signaling and Ca2+-transporting ATPases

“…Up to this point, Pmr1 was the only SPCA member to be extensively characterized. Inactivation of PMR1 resulted in missorting and misprocessing of proteins along the secretory pathway and to N-and O-linked glycosylation defects (3,25). These observations were consistent with depletion of luminal Golgi stores and could be separately corrected by high levels of extracellular Ca 2ϩ and Mn 2ϩ , respectively (25).…”

“…Inactivation of PMR1 resulted in missorting and misprocessing of proteins along the secretory pathway and to N-and O-linked glycosylation defects (3,25). These observations were consistent with depletion of luminal Golgi stores and could be separately corrected by high levels of extracellular Ca 2ϩ and Mn 2ϩ , respectively (25). In addition, pmr1 mutants have elevated resting levels of Ca 2ϩ in the cytosol and cannot effectively maintain cellular Ca 2ϩ and Mn 2ϩ homeostasis (33,35,62).…”

“…Yeast Pmr1 (for "plasma membrane ATPase related") was the first member of the family of secretory pathway Ca 2ϩ -ATPases (SPCA) to be identified (71). It mediates highaffinity Ca 2ϩ (and Mn 2ϩ ) transport under normal physiological conditions and serves a dual role in maintaining cytosolic ion homeostasis as well as in supplying the Golgi lumen with Ca 2ϩ and Mn 2ϩ for protein sorting, processing, and glycosylation (25). Pmr1 homologs are now known to be ubiquitous in all higher eukaryotes, with the notable exception of plants, and much of what we know about the structure and physiological role of these transporters comes from studies in yeast.…”

Functional expression of mung bean Ca2+/H+ antiporter in yeast and its intracellular localization in the hypocotyl and tobacco cells

“…Organelle-specific Ca 21 ATPases have also been reported for plants [3,33±35]. It has recently been reported that several processes in the Golgi lumen are Ca 21 dependent in mammalian and yeast cells [36,37,39,40]. Therefore, these two active Ca 21 transporters may keep the Ca 21 concentration in the Golgi lumen at high levels.…”

Characterization of a cDNA from Beta maritima that confers nickel tolerance in yeast

“…The endoplasmic reticulum, Golgi, and small vesicles detoxify metals by removing them from the cytoplasm (Durr et al, 1998;Wu et al, 2002). For example, the Eca1 protein located in the endoplasmic reticulum leads to the uptake of manganese and confers manganese tolerance to the cells (Wu et al, 2002).…”