In eukaryotic cells, COPI vesicles retrieve resident proteins to the endoplasmic reticulum and mediate intra-Golgi transport. Here, we studied the Hansenula polymorpha homologue of the Saccharomyces cerevisiae RET1 gene, encoding ␣-COP, a subunit of the COPI protein complex. H. polymorpha ret1 mutants, which expressed truncated ␣-COP lacking more than 300 C-terminal amino acids, manifested an enhanced ability to secrete human urokinase-type plasminogen activator (uPA) and an inability to grow with a shortage of Ca 2؉ ions, whereas a lack of ␣-COP expression was lethal. The ␣-COP defect also caused alteration of intracellular transport of the glycosylphosphatidylinositol-anchored protein Gas1p, secretion of abnormal uPA forms, and reductions in the levels of Pmr1p, a Golgi Ca 2؉ -ATPase. Overexpression of Pmr1p suppressed some ret1 mutant phenotypes, namely, Ca 2؉ dependence and enhanced uPA secretion. The role of COPI-dependent vesicular transport in cellular Ca 2؉ homeostasis is discussed.In eukaryotic cells, secretory proteins synthesized at the endoplasmic reticulum (ER) pass through multiple distinct membrane-bound organelles comprising the secretory pathway. Transport of proteins and lipids between membrane compartments of the early secretory system is mediated by COPIand COPII-coated vesicles that capture cargo, bud from the donor membrane, and then target, dock, and fuse with an appropriate acceptor compartment (40). Both the COPI and COPII protein coat complexes employ a GTP switch mechanism for coating and uncoating. The COPII complex mediates selective protein export from the ER, while COPI-coated vesicles retrieve resident proteins to the ER and mediate intraGolgi transport (8,38).The Golgi apparatus is composed of distinct cisternal regions, namely, the cis, medial, trans-Golgi, and trans-Golginetwork regions (13). Secretory cargo proteins passing through the Golgi apparatus are modified by the unique sets of resident enzymes of different subcompartments. Two alternative models for the origin of the Golgi subcompartments suggest different roles for COPI vesicles in secretory cargo transport and the function of the Golgi apparatus. In the stable-compartments model, anterograde COPI vesicles carry secretory cargo forward and exclude resident Golgi proteins. In the cisternalmaturation model, entire cisternae carry secretory cargo forward, and retrograde COPI vesicles recycle resident Golgi proteins to younger cisternae. Thus, the distinction between the two models centers on the contents of COPI vesicles and the directionality of their transport. However, recent experimental data indicate that intra-Golgi COPI traffic has a bidirectional character (for reviews, see references 23 and 38). Moreover, the dynamics of cisternal progression and vesicular traffic are different in higher eukaryotes versus yeasts. Progression of the tightly stacked Golgi cisternae of animal cells occurs at a significantly lower rate than the traffic of most cargoes through the secretory pathway. COPI vesicles mediating cargo tra...