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 Mn(2+) 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 Mn(2+) in ∆spf1 cells and an increase following it's overexpression. In agreement with the observed loss of luminal Mn(2+) we could observe concurrent reduction in many Mn(2+)-related process in the ER lumen. Conversely, cytosolic Mn(2+)-dependent processes were increased. Together, these data support a role for Spf1p in Mn(2+) 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 Mn(2+)-dependent neurological disorders.

Figure 1. Spf1 affects the cellular distribution of Mn²⁺.

(A) Metal content of microsomes from Δspf1, OE-Spf1 and WT yeast was determined using ICP-mass spectrometry. By looking at the fold change of ion concentration of Δspf1 or OE-Spf1 microsomes relative to WT it can be seen that Mn²⁺ is the only ion that decreases in Δspf1 and increases in microsomes of OE-Spf1. (B) Representative fluorescent images of cells expressing GFP tagged Smf1 or Smf2 proteins which act in the uptake and intracellular trafficking of Mn²⁺. In Δspf1 these reporters of luminal Mn²⁺ are stabilized and can be seen on the cell surface or intracellular vesicles and less in the vacuole. Scale bar: 5µm. (C) Intensity measurements by flow cytometry of GFP tagged Smf1 or Smf2 proteins indicate higher levels of these proteins in ∆spf1 cells compared to WT. *** P value<0.001.

Figure 2. Biosynthesis of sphingolipids is interrupted due to deletion of SPF1.

(A) WT and Δspf1 cells were plated in serial dilution on YPD plates without and with Aureobasidin A, an IPC synthase inhibitor. Loss of SPF1 increases the sensitivity to this inhibitor. (B) The levels of different types of IPC, were measured by mass spectrometry and are mostly lower in Δspf1 cells compared to WT. (C) The levels of different types of MIPC and M(IP)2C, were measured by mass spectrometry and are mostly lower in Δspf1 cells compared to WT. * P value<0.05, ** P value<0.01, *** P value<0.001.

Figure 3. Δspf1 cells are more sensitive to the ergosterol biosynthesis inhibitor Terbinafine and display change in ergosterol distribution compared to WT.

(A) WT and Δspf1 cells were plated in serial dilution on YPD plates without and with Terbinafine, an ergosterol biosynthesis inhibitor. Loss of SPF1 causes growth inhibition in the presence of this inhibitor. (B) WT and Δspf1 cells were stained for sterol distribution by fillipin staining. While most of the Δspf1 cells exhibit homogeneous sterol distribution, the WT shows a non-homogenous sterol staining pattern. Scale bar: 5µm.

Figure 4. Spf1 affects the exit of GPI anchor proteins from the ER.

WT and ∆spf1 cells expressing tagged GPI anchored proteins (YFP-Ccw14 and RFP-Gas1) were imaged. While these proteins are normally localized to the cell periphery and the vacuole, a deletion of SPF1 enhances ER retention. Scale bar: 5µm.

Figure 5. Spf1 affects protein mannosylation and its ATPase activity is required for its molecular function.

(A) Western blot of three GPI anchored proteins that undergo known mannosylation (Gas1, Ccw14 and Yps1) reveals that this modification is reduced in Δspf1 compared to WT. (B) Western blot of Gas1 in WT and Δspf1 cells without any plasmid, with a plasmid containing full-length SPF1 gene, or with plasmid containing the full-length gene carrying a mutation rendering the protein ATPase-dead. Lack of rescue in the ATPase dead mutant confirms that the reason for the defect in Gas1 maturation is the absence of the functional ATPase activity of Spf1.

Figure 6. ATP13A1 is the functional homologue of Spf1.

(A) Immunostaining of HeLa cells with α-ATP13A1 and DAPI (nucleus staining) indicates perinuclear localization for ATP13A1. Scale bar: 10µm. (B) Amount of spliced XBP1 (a result of ER stress) was assayed by qPCR analysis. Silencing of ATP13A1 in HeLa cells causes an increase in the spliced form. (C) Western blot of BiP protein reveals that its amount is increased upon silencing of ATP13A1 in HeLa cells. Blot was also probed with an antibody against GAPDH as a loading control. (D) Mass Spectrometry analysis reveals a specific increase in GlcCer levels in cells silenced for ATP13A1 suggesting enhanced activity of GlcCer synthase. (E) Fluorescent cholera toxin subunit B (CT-B) was used to stain the plasma membrane ganglioside GM1. Cells treated with an siRNA against ATP13A1 display accumulation of GM1. Scale bar: 10µm.