Abstract
The Schizosaccharomyces pombe shu1+ gene encodes a cell-surface protein required for assimilation of exogenous heme. In this study, shaving experiments showed that Shu1 is released from membrane preparations when spheroplast lysates are incubated with phosphoinositide-specific phospholipase C (PI-PLC). Shu1 cleavability by PI-PLC and its predicted hydropathy profile strongly suggested that Shu1 is a glycosylphosphatidylinositol-anchored protein. When heme biosynthesis is selectively blocked in hem1Δ mutant cells, the heme analog zinc mesoporphyrin IX (ZnMP) first accumulates into vacuoles and then subsequently, within the cytoplasm in a rapid and Shu1-dependent manner. An HA4-tagged shu1+ allele that retained wild-type function localizes to the cell surface in response to low hemin concentrations, but under high hemin concentrations, Shu1-HA4 re-localizes to the vacuolar membrane. Inactivation of abc3+, encoding a vacuolar membrane transporter, results in hem1Δ abc3Δ mutant cells being unable to grow in the presence of hemin as the sole iron source. In hem1Δ abc3Δ cells, ZnMP accumulates primarily in vacuoles and does not sequentially accumulate in the cytosol. Consistent with a role for Abc3 as vacuolar hemin exporter, results with hemin-agarose pulldown assays showed that Abc3 binds to hemin. In contrast, an Abc3 mutant in which an inverted Cys-Pro motif had been replaced with Ala residues fails to bind hemin with high affinity. Taken together, these results show that Shu1 undergoes rapid hemin-induced internalization from the cell surface to the vacuolar membrane and that the transporter Abc3 participates in the mobilization of stored heme from the vacuole to the cytosol.
Highlights
The Schizosaccharomyces pombe shu1؉ gene encodes a cellsurface protein required for assimilation of exogenous heme
An Abc3 mutant in which an inverted Cys-Pro motif had been replaced with Ala residues fails to bind hemin with high affinity. These results show that Shu1 undergoes rapid hemininduced internalization from the cell surface to the vacuolar membrane and that the transporter Abc3 participates in the mobilization of stored heme from the vacuole to the cytosol
Shu1 and Abc3 Are Involved in Heme Acquisition this three-dimensional structure of a CFEM protein, disulfide bonds formed by the 8 conserved Cys residues are predicted to stabilize the helical basket-fold, which itself would serve as a flat platform in which a planar heme molecule would sit at the top of the structure
Summary
Shu Is a Membrane Protein That Exhibits PI-PLC Sensitivity—Microscopic analysis using spheroplasts have previously shown that Shu is a plasma membrane protein [25]. Consistent with iron-mediated repression of abc3ϩ-GFP and shu1ϩ-HA4 mRNA levels (under the control of abc3ϩ and shu1ϩ promoters, respectively), Abc3-GFP and Shu1-HA4 proteins were barely or not detected in membrane protein fractions that had been prepared from iron-replete cells (Fig. 7) Taken together, these results showed that Abc expressed in S. pombe interacts with hemin, supporting the proposed model that Abc is required as a means to mobilize stored heme from the vacuole to the cytoplasm. In the case of cells lacking Shu and harboring an empty plasmid, they failed to accumulate the ZnMP fluorescent compound in comparison with hem1⌬ shu1⌬ abc3ϩ cells expressing a wild-type shu1ϩ that was re-integrated (Fig. 9A). These results suggested that mobilization of a heme analog (ZnMP) from the vacuole to the cytosol requires Abc and involves an inverted CP motif (residues 151–152) within its amino-terminal region
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