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

Abstract

In Saccharomyces cerevisiae, ARN1 encodes a transporter for the uptake of ferrichrome, an important nutritional source of iron. In the absence of ferrichrome, Arn1p is sorted directly from the trans-Golgi network (TGN) to the vacuolar lumen via the vacuolar protein-sorting pathway. Arn1p is mis-sorted to the plasma membrane in cells lacking Gga2p, a monomeric clathrin-adaptor protein involved in vesicular transport from the TGN. Although Ggas have been characterized as ubiquitin receptors, we show here that ubiquitin binding by Gga2 was not required for the TGN-to-endosome trafficking of Arn1, but it was required for subsequent sorting of Arn1 into the multivesicular body. In a ubiquitin-binding mutant of Gga2, Arn1p accumulated on the vacuolar membrane in a ubiquitinated form. The yeast epsins Ent3p and Ent4p were also involved in TGN-to-vacuole sorting of Arn1p. Amino-terminal sequences of Arn1p were required for vacuolar protein sorting, as mutation of ubiquitinatable lysine residues resulted in accumulation on the vacuolar membrane, and mutation of either a THN or YGL sequence resulted in mis-sorting to the plasma membrane. These studies suggest that Gga2 is involved in sorting at both the TGN and multivesicular body and that the first step can occur without ubiquitin binding.

Direct binding of the Kex2p cytosolic tail to the VHS domain of yeast Gga2p facilitates TGN to prevacuolar compartment transport and is regulated by phosphorylation

“…In addition, several studies suggest that sorting of the polytopic membrane proteins Gap1p, Fur4p, Sit1p, and Arn1p involves interaction of covalently bound ubiquitin with the GAT domains of the Gga proteins Scott et al, 2004;Kim et al, 2007), leading to the notion that yeast Ggas might exclusively mediate sorting of ubiquitin-modified cargo. Recent work, however, suggested that TGN-PVC trafficking of Arn1p and Gap1p, albeit Gga dependent, is independent of both ubiquitin modification and the Gga GAT domain, although the mechanism is unknown (Deng et al, 2009;Lauwers et al, 2009). Although the C-tails of both Kex2p and Vps10p are highly acidic, they contain no obvious acidic dileucine motifs.…”

“…Recent evidence suggests that the ubiquitin-GAT interaction may be required only for Gga-dependent sorting at the PVC into the luminal vesicles of multivesicular endosomes and that other interactions mediate Gga-dependent sorting of polytopic proteins at the TGN (Deng et al, 2009;Lauwers et al, 2009). Whether the ubiquitin-independent interactions at the TGN involve direct binding to the Gga VHS domains remains to be seen.…”

Metabolic Response to Iron Deficiency in Saccharomyces cerevisiae

“…Fractions were collected and subjected to SDS-PAGE and Western blotting using peroxidase anti-peroxidase, anti-GFP (Roche Applied Science) at 1:1000, and anti-Dpm1 (Molecular Probes) at 1:1000. Immunoprecipitations from 55 Fe-labeled cells were performed as described (27) using IgG-Sepharose beads (Sigma). Immune complexes were washed, and 10% of the total washed beads were used for Western blotting, whereas the remainder was subjected to scintillation counting.…”

Functional characterization of protein-sorting machineries at the trans-Golgi network in Drosophila melanogaster

“…dGGA can suppress the defect in the vacuolar protein sorting of yeast gga1gga2 double disruptant In yeast, Gga proteins have been well characterized and are known to play important roles in sorting of membrane proteins in the TGNto-endosomes routes (Hirst et al, 2000;Dell'Angelica et al, 2000;Black and Pelham, 2000;Costaguta et al, 2001;Scott et al, 2004;Deng et al, 2009). The yeast mutant lacking both GGA genes is defective in both sorting of vacuolar soluble proteins such as CPY and processing of mating pheromones (Hirst et al, 2000;Dell'Angelica et al, 2000;Black and Pelham, 2000;Costaguta et al, 2001).…”

“…In the Gga-deficient cells, missorting of vacuolar proteinases such as carboxypeptidase Y (CPY) to the extracellular space occurs due to a defect in the transport of the sorting receptor Vps10p that shuttles between the TGN and endosomes (Hirst et al, 2000;Dell'Angelica et al, 2000). The yeast Ggas are also involved in the Golgi-to-vacuole trafficking of ubiquitylated cargo molecules, such as the Gap1 and Fur4 permeases and of the ferrichrome transporter Arn1p, at the transport step of these membrane proteins into the internal vesicles of multivesicular bodies (MVB) (Scott et al, 2004;Deng et al, 2009;Lauwers et al, 2009).…”