The Yeast P5 Type ATPase, Spf1, Regulates Manganese Transport into the Endoplasmic Reticulum

Abstract

The endoplasmic reticulum (ER) is a large, multifunctional and essential organelle. Despite intense research, the function of more than a third of ER proteins remains unknown even in the well-studied model organism Saccharomyces cerevisiae. One such protein is Spf1, which is a highly conserved, ER localized, putative P-type ATPase. Deletion of SPF1 causes a wide variety of phenotypes including severe ER stress suggesting that this protein is essential for the normal function of the ER. The closest homologue of Spf1 is the vacuolar P-type ATPase Ypk9 that influences Mn2+ homeostasis. However in vitro reconstitution assays with Spf1 have not yielded insight into its transport specificity. Here we took an in vivo approach to detect the direct and indirect effects of deleting SPF1. We found a specific reduction in the luminal concentration of Mn2+ in ∆spf1 cells and an increase following it’s overexpression. In agreement with the observed loss of luminal Mn2+ we could observe concurrent reduction in many Mn2+-related process in the ER lumen. Conversely, cytosolic Mn2+-dependent processes were increased. Together, these data support a role for Spf1p in Mn2+ transport in the cell. We also demonstrate that the human sequence homologue, ATP13A1, is a functionally conserved orthologue. Since ATP13A1 is highly expressed in developing neuronal tissues and in the brain, this should help in the study of Mn2+-dependent neurological disorders.

Inhibition of the Formation of the Spf1p Phosphoenzyme by Ca2+

“…The levels of EP were slightly decreased by Mn 2ϩ , suggesting that Mn 2ϩ may substitute Ca 2ϩ with lower efficiency. However, we have previously found that Mn 2ϩ decreases Spf1p ATPase activity (18), a result that does not support the proposed role of Spf1p as a Mn 2ϩ transporter (28). The ATP dependence of the phosphorylation reaction indicates that Spf1p reacts with ATP with high affinity, as expected for the catalytic ATP site of a P-ATPase, and that it was not significantly changed by Ca 2ϩ .…”

Endoplasmic reticulum stress and calcium imbalance are involved in cadmium-induced lipid aberrancy in Saccharomyces cerevisiae

“…This data supports that PEMT pathway is also essential for mitochondrial function during ER stress and impairment of calcium. The defect of Pmr1p and Spf1p, implicated in protein glycosylation, influenced the conditions of ER stress stimulates LD formation in Saccharomyces cerevisiae (Fei et al 2009;Cohen et al 2013). Besides, the role of LD biogenesis and synthesis of TAG is a sign of cellular stress, and it was proposed that alterations in PC homeostasis might be the source of TAG (Al-Saffar et al 2002 andIorio et al 2003).…”

Manganese Redistribution by Calcium-stimulated Vesicle Trafficking Bypasses the Need for P-type ATPase Function

“…Spf1 and Smf2 activities might be required for ER and Golgi manganese supply and thus be required for vesicle-mediated manganese transport. Recently, the Spf1 has been shown to regulate Mn 2ϩ transport into the ER (14), and the addition of extracellular Ca 2ϩ accordingly suppressed SPF1 mutant phenotypes (35,55). Smf2 was predicted to transport Mn 2ϩ across membranes toward the cytosol by the assumption that Nramp transporters transport divalent cations in this direction (8).…”

“…B, CaCl 2 fails to rescue CFW resistance of pmr1⌬ mutants in the absence of Spf1 or Smf2. Shown is CFW sensitivity of pmr1⌬ upon additional deletion of the low affinity Mn 2ϩ transporter PHO84 (12), the vesicular Mn 2ϩ transporter SMF2 (10), the vacuolar Ca 2ϩ /H ϩ exchanger VCX1 (68), the plasma membrane Ca 2ϩ channel CCH1 (69), the vacuolar Fe 2ϩ /Mn 2ϩ transporter CCC1 (19), and the putative ER Mn 2ϩ -transporter SPF1 (14). Drop test analysis of WT, pmr1⌬, pho84⌬, pmr1⌬ pho84⌬, smf2⌬, pmr1⌬ smf2⌬, vcx1⌬, pmr1⌬ vcx1, cch1, pmr1⌬ cch1⌬, ccc1⌬, pmr1⌬ccc1⌬, spf1⌬, and pmr1⌬ spf1⌬ cells is shown.…”

“…Atx2 localizes to Golgi-like vesicles, but the mechanism by which Atx2 regulates intracellular Mn 2ϩ levels remains unknown (13). Recently, the P-type ATPase Spf1 (hATP13A1) has been suggested to regulate Mn 2ϩ transport into the endoplasmic reticulum (ER) (14), whereas Pmr1, a Golgi-localized P-type Ca 2ϩ and Mn 2ϩ ATPase, pumps cytosolic Mn 2ϩ into the lumen of the Golgi (15)(16)(17). Apart from providing sugar transferases with Mn 2ϩ as cofactor, Pmr1 has another role in Mn 2ϩ detoxification by secretory pathway-mediated excretion (16 -18).…”