PMR1, a Ca2+-ATPase in Yeast Golgi, Has Properties Distinct from Sarco/endoplasmic Reticulum and Plasma Membrane Calcium Pumps

doi:10.1074/jbc.272.15.9895

PMR1, a Ca²⁺-ATPase in Yeast Golgi, Has Properties Distinct from Sarco/endoplasmic Reticulum and Plasma Membrane Calcium Pumps*

From the Department of Physiology, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205

^‡ To whom correspondence should be addressed:
Dept. of Physiology, The Johns Hopkins University School of Medicine, 725 N. Wolfe St., Baltimore
, MD 21205. Tel.: 410-955-4732; Fax: 410-955-0461; E-mail: rajini_rao{at}qmail.bs.jhu.edu

Abstract

PMR1, a P-type ATPase cloned from the yeast Saccharomyces cerevisiae, was previously localized to the Golgi, and shown to be required for normal secretory processes (Antebi, A., and Fink, G.R. (1992) Mol. Biol. Cell 3, 633-654). We provide biochemical evidence that PMR1 is a Ca²⁺-transporting ATPase in the Golgi, a hitherto unusual location for a Ca²⁺ pump. As a starting point for structure-function analysis using a mutagenic approach, we used the strong and inducible heat shock promoter to direct high level expression of PMR1 from a multicopy plasmid. Yeast lysates were separated on sucrose density gradients, and fractions assayed for organellar markers. PMR1 is found in fractions containing the Golgi marker guanosine diphosphatase, and is associated with an ATP-dependent, protonophore-insensitive ⁴⁵Ca²⁺ uptake activity. This activity is virtually abolished in the absence of the expression plasmid. Furthermore, replacement of the active site aspartate within the phosphorylation domain had the expected effect of abolishing Ca²⁺ transport activity entirely. Interestingly, the mutant enzymes (Asp-371 → Glu and Asp-371 → Asn) demonstrated proper targeting to the Golgi, unlike analogous mutations in the related yeast H⁺-ATPase. Detailed characterization of calcium transport by PMR1 showed that sensitivity to inhibitors (vanadate, thapsigargin, and cyclopiazonic acid) and affinity for substrates (MgATP and Ca²⁺) were different from the previously characterized sarco/endoplasmic reticulum and plasma membrane Ca²⁺-ATPases. PMR1 therefore represents a new and distinct P-type Ca²⁺-ATPase. Because close homologs of PMR1 have been cloned from rat and other organisms, we suggest that Ca²⁺-ATPases in the Golgi will form a discrete subgroup that are important for functioning of the secretory pathway.

Footnotes

↵* This work was supported by Grants IRG11-33 and JFRA 538 from the American Cancer Society, Grant-in-aid 95012290 from the American Heart Association, and Grant GM52414 from the National Institutes of Health (to R. R.). The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

SERCA

sarco/endoplasmic reticulum Ca²⁺-ATPase

CCCP

carbonyl cyanide m-chlorophenylhydrazone

Mes

4-morpholineethanesulfonic acid

PAGE

polyacrylamide gel electrophoresis

TGN

trans-Golgi network

PMCA

plasma membrane; Ca²⁺-ATPase.
↵² V. Marchi, A. Sorin, and R. Rao, unpublished observations.
↵³ Y. Song, personal communication.
↵⁴ R. K. Nakamoto, S. Verjovski-Almeida, K. Allen, A. Ambesi, R. Rao, and C. W. Slayman, manuscript in preparation.
- Received December 4, 1996.
- Revision received February 6, 1997.