Abstract

We have studied the intracellular trafficking of Sit1 [ferrioxamine B (FOB) transporter] and Enb1 (enterobactin transporter) in Saccharomyces cerevisiae using green fluorescent protein (GFP) fusion proteins. Enb1 was constitutively targeted to the plasma membrane. Sit1 was essentially targeted to the vacuolar degradation pathway when synthesized in the absence of substrate. Massive plasma membrane sorting of Sit1 was induced by various siderophore substrates of Sit1, and by coprogen, which is not a substrate of Sit1. Thus, different siderophore transporters use different regulated trafficking processes. We also studied the fate of Sit1-mediated internalized siderophores. Ferrioxamine B was recovered in isolated vacuolar fractions, where it could be detected spectrophotometrically. Ferrioxamine B coupled to an inhibitor of mitochondrial protoporphyrinogen oxidase (acifluorfen) could not reach its target unless the cells were disrupted, confirming the tight compartmentalization of siderophores within cells. Ferrioxamine B coupled to a fluorescent moiety, FOB-nitrobenz-2-oxa-1,3-diazole, used as a Sit1-dependent iron source, accumulated in the vacuolar lumen even in mutants displaying a steady-state accumulation of Sit1 at the plasma membrane or in endosomal compartments. Thus, the fates of siderophore transporters and siderophores diverge early in the trafficking process.

Highlights

  • Re-use of this article is permitted in accordance with the Creative Commons Deed, Attribution 2.5, which does not permit commercial exploitation

  • Differences in the behaviour of the four siderophore transport systems of S. cerevisiae We checked whether the four siderophore transporters of S. cerevisiae (Arn1, Enb1, Sit1 and Taf1) displayed similar intracellular trafficking – sorting from the endosomes to plasma membrane triggered by a specific substrate

  • The Sit1-green fluorescent protein (GFP), Arn1-GFP and Enb1-GFP transporters were detected in cells grown in both iron-rich (YPD) and iron-deficient (YPD þ bathophenanthroline disulfonic acid (BPS)) conditions (Figure 2A), they were more abundant under iron-deficient growth conditions, as shown by fluorescence and western immunoblotting (Figure 2B)

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Summary

Introduction

Re-use of this article is permitted in accordance with the Creative Commons Deed, Attribution 2.5, which does not permit commercial exploitation. The siderophore pathway mediates iron uptake from siderophores – small molecules that bind, solubilize and chelate ferric iron in the environment with high affinity (reviewed in 11). They are synthesized in a non-ribosomal enzymatic process and secreted by bacteria and fungi, but not by S. cerevisiae. It has been shown that Arn1 [carrying a C-terminal hemagglutinin (HA) tag] is sorted directly from the Golgi to the endosomal compartment and is not sorted to the plasma membrane unless the cells are exposed to low concentrations of FCH [19], which acts on a high-affinity C-terminal receptor domain of the protein to control intracellular trafficking of the transporter [20]. We used strains bearing green fluorescent protein (GFP)tagged versions of the siderophore transporters under the control of the endogenous promoter or under the control of a galactose-inducible promoter (pGAL) to explore potential substrate-induced regulation of the trafficking of these two siderophore transporters

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