Abstract
In the fission yeast Schizosaccharomyces pombe, acquisition of exogenous heme is largely mediated by the cell membrane-associated Shu1. Here, we report that Str3, a member of the major facilitator superfamily of transporters, promotes cellular heme import. Using a strain that cannot synthesize heme de novo (hem1Δ) and lacks Shu1, we found that the heme-dependent growth deficit of this strain is rescued by hemin supplementation in the presence of Str3. Microscopic analyses of a hem1Δ shu1Δ str3Δ mutant strain in the presence of the heme analog zinc mesoporphyrin IX (ZnMP) revealed that ZnMP fails to accumulate within the mutant cells. In contrast, Str3-expressing hem1Δ shu1Δ cells could take up ZnMP at a 10-μm concentration. The yeast Saccharomyces cerevisiae cannot efficiently transport exogenously supplied hemin. However, heterologous expression of Str3 from S. pombe in S. cerevisiae resulted in ZnMP accumulation within S. cerevisiae cells. Moreover, hemin-agarose pulldown assays revealed that Str3 binds hemin. In contrast, an Str3 mutant in which Tyr and Ser residues of two putative heme-binding motifs (530YX3Y534 and 552SX4Y557) had been replaced with alanines exhibited a loss of affinity for hemin. Furthermore, this Str3 mutant failed to rescue the heme-dependent growth deficit of a hem1Δ shu1Δ str3Δ strain. Further analysis by absorbance spectroscopy disclosed that a predicted extracellular loop region in Str3 containing the two putative heme-binding motifs interacts with hemin, with a KD of 6.6 μm Taken together, these results indicate that Str3 is a second cell-surface membrane protein for acquisition of exogenous heme in S. pombe.
Highlights
In the fission yeast Schizosaccharomyces pombe, acquisition of exogenous heme is largely mediated by the cell membrane– associated Shu[1]
Str[3] is a heme transporter in fission yeast presence of 0.15 M hemin, results showed that these strains these results revealed that Str[3] supports hemin acquisition in exhibited robust growth in a manner similar to hem1⌬ shu1⌬ the presence of 0.15 M hemin, but it is not effective when cells expressing an endogenous str3ϩ (Fig. 3C)
In the case of the major facilitator superfamily (MFS)-type iron-siderophore transporter, loop 11 contains the siderophore transporter domain, in which a highly conserved Tyr residue is present and required for uptake of siderophore into the cells (23). This highly conserved Tyr residue was found in Str[3], loop 11 of Str[3] was unique due to the presence of two putative heme-binding sequences, 530YX3Y534 and 552SX4Y557, that were absent in S. pombe Str[1] and Str[2] proteins. These two putative heme-binding sequences were reminiscent of neariron transporter (NEAT) motifs that are found in several Grampositive bacteria (24)
Summary
In vital biochemical processes, such as respiration, oxygen transport, and disproportionation of hydrogen peroxide, respectively (2). In vivo functional growth assays have shown that the presence of Shu[1] is required for assimilation of exogenous hemin by S. pombe hem1⌬ cells (7). When heme biosynthesis is selectively blocked in hem1⌬ mutant cells, ZnMP first accumulates into vacuoles and within the cytosol (8) Consistent with this observation, results have shown that in response to elevated concentrations of hemin or ZnMP, Shu[1] undergoes internalization from the cell surface to the vacuole (8). In the case of Str[1], its heterologous expression in a Saccharomyces cerevisiae mutant strain that is deficient in high-affinity iron uptake systems results in assimilation of iron from ferrichrome (11) This result is consistent with the fact that S. pombe possesses the ability to produce ferrichrome (14) and with the plasma membrane localization of Str[1] in S. pombe. These results are consistent with the existence of an additional hemin acquisition pathway in S. pombe that involves the action of Str[3]
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