Trafficking of Siderophore Transporters in Saccharomyces cerevisiae and Intracellular Fate of Ferrioxamine B Conjugates

Abstract

†These authors contributed equally to this study.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.

Response to Iron Deprivation in Saccharomyces cerevisiae

“…Arn1p is then ubiquitinated by Rsp5p and sorted into the multivesicular body for delivery to the vacuolar lumen. Both Arn1p and Arn3p/Sit1p are diverted from the vacuolar sorting pathway to the plasma membrane in the presence of their respective siderophore substrates, and in the case of Arn1p, this relocalization involves the binding of ferrichrome to a receptor domain of the Arn1p transporter itself (14,22). In contrast, Arn4p/Enb1p traffics directly to the cell surface, even in the absence of its substrate, enterobactin.…”

“…Both ubiquitination via Rsp5p and the activity of Gga1p and -2p (C. C. Philpott and Y. Deng, unpublished observations) are involved in the internalization of Arn1p. Although mutations that inhibit actin-dependent endocytosis and the cycling of Arn1p also inhibit the uptake of ferrichrome, mutations in Gga2p and Rsp5, which inhibit cycling of Arn1p and Arn3p without interfering with the endocytosis machinery, do not inhibit uptake of siderophores, indicating that cycling is not required for uptake (14,21). The internalization of the transporters to a sorting compartment such as the early endosome may allow the cell to selectively recycle the transporters to the plasma membrane for continued uptake or divert them to the late endosomal pathway for vacuolar degradation.…”

“…After uptake through Arn3/Sit1p, intracellular ferrioxamine B accumulates in the vacuole as an intact chelate (14). Whether the iron dissociates from the siderophore within the vacuole or the chelate is transported to the cytosol prior to dissociation is not known.…”

“…Some of the siderophore transporters are regulated posttranslationally by their localization within the late secretory pathway. Both Arn1p and Arn3p/Sit1p traffic directly from the trans-Golgi network to the vacuole for degradation when their respective siderophore substrates are not present extracellularly (14,23). Arn1p is recognized at the trans-Golgi network by Gga2p, a a Ferrichromes include ferrichrome, ferrichrome A, ferricrocin, ferrichrycin, ferrirhodin, and ferrirubin.…”

Gga2 Mediates Sequential Ubiquitin-independent and Ubiquitin-dependent Steps in the Trafficking of ARN1 from the trans-Golgi Network to the Vacuole

“…Unlike Arn1 and Arn3, Arn4 is expressed exclusively on the plasma membrane, whether enterobactin is present in the growth medium or not (Fig. 5B) (48). We constructed a transporter that contained the Arn1 amino-terminal and transmembrane domains fused to the carboxyl-terminal domain of Arn4 (Arn1-N,M/ Arn4-C).…”