Heme acquisition by Shu1 requires Nbr1 and proteins of the ESCRT complex in Schizosaccharomyces pombe.

ABSTRACT

Assimilation of heme is mediated by the cell surface protein Shu1 in Schizosaccharomyces pombe. Shu1 undergoes internalization from the cell surface to the vacuole in response to high concentrations of hemin. Here, we have identified cellular components that are involved in mediating vacuolar targeting of Shu1. Cells deficient in heme biosynthesis and lacking the polyubiquitin gene ubi4+ exhibit poor growth in the presence of exogenous hemin as a sole source of heme. Microscopic analyses of hem1Δ shu1Δ ubi4Δ cells expressing a functional HA4 -tagged Shu1 show that Shu1 localizes to the cell surface. Ubiquitinated Nbr1 functions as a receptor for the endosomal sorting complexes required for transport (ESCRT) that delivers cargos to the vacuole. Inactivation of nbr1+ , ESCRT-0 hse1+ or ESCRT-I sst6+ results in hem1Δ cells being unable to use exogenous hemin for the growth. Using lysate preparations from hemin-treated cells, Shu1-Nbr1 and Shu1-Hse1 complexes are detected by coimmunoprecipitation experiments. Further analysis by immunofluorescence microscopy shows that Shu1 is unable to reach vacuoles of hemin-treated cells harboring a deletion for one of the following genes ubi4+ , nbr1+ , hse1+ and sst6+ . Together, these results reveal that hemin-mediated vacuolar targeting of Shu1 requires Ubi4-dependent ubiquitination, the receptor Nbr1 and the ESCRT proteins Hse1 and Sst6.