Greg Odorizzi

Receptor downregulation and multivesicular-body sorting.

The sorting of proteins into the inner vesicles of multivesicular bodies is required for many key cellular processes, which range from the downregulation of activated signalling receptors to the proper stimulation of the immune response. Recent advances in our understanding of the multivesicular-body sorting pathway have resulted from the identification of ubiquitin as a signal for the efficient sorting of proteins into this transport route, and from the discovery of components of the sorting and regulatory machinery that directs this complex process.

Fab1p PtdIns(3)P 5-Kinase Function Essential for Protein Sorting in the Multivesicular Body

Sorting of signal-transducing cell surface receptors within multivesicular bodies (MVBs) is required for their rapid down-regulation and degradation within lysosomes. Yeast mutants defective in late stages of transport to the vacuole/lysosome accumulate MVBs. We demonstrate that the membrane glycoprotein carboxypeptidase S and the G protein-coupled receptor Ste2p are targeted into the vacuole lumen, and this process requires a subset of VPS gene products essential for normal endosome function. The PtdIns(3)P 5-kinase activity of Fab1p, which converts the product of the Vps34p PtdIns 3-kinase PtdIns(3)P into PtdIns(3,5)P2, also is required for cargo-selective sorting into the vacuole lumen. These findings demonstrate a role for phosphoinositide signaling at distinct stages of vacuolar/lysosomal protein transport and couple PtdIns(3,5)P2 synthesis to regulation of MVB sorting.

Mammalian tumor susceptibility gene 101 (TSG101) and the yeast homologue, Vps23p, both function in late endosomal trafficking

The mammalian tumor susceptibility gene tsg101 encodes the homologue of Vps23p, a class E Vps protein essential for normal membrane trafficking in the late endosome/multivesicular body of yeast. Both proteins assemble into large (∼350 kDa) cytosolic protein complexes and we show that the yeast complex contains another class E Vps protein, Vps28p. tsg101 mutant cells exhibit defects in sorting and proteolytic maturation of the lysosomal hydrolase cathepsin D, as well as in the steady‐state distribution of the mannose‐6‐phosphate receptor. Additionally, endocytosed EGF receptors that are normally sorted to the lysosome are instead rapidly recycled back to the cell surface in tsg101 mutant cells. We propose that tsg101 mutant cells are defective in the delivery of cargo proteins to late endosomal compartments. One consequence of this endosomal trafficking defect is the delayed down‐regulation/degradation of activated cell surface receptors, resulting in prolonged signaling. This may contribute to the tumorigenic phenotype exhibited by the tsg101 mutant fibroblasts.

The AP-3 Adaptor Complex Is Essential for Cargo-Selective Transport to the Yeast Vacuole

Three distinct adaptor protein (AP) complexes involved in protein trafficking have been identified. AP-1 and AP-2 mediate protein sorting at the trans-Golgi network and plasma membrane, respectively, whereas the function of AP-3 has not been defined. A screen for factors specifically involved in transport of alkaline phosphatase (ALP) from the Golgi to the vacuole/lysosome has identified Ap16p and Ap15p of the yeast AP-3 complex. Deletion of each of the four AP-3 subunits results in selective mislocalization of ALP and the vacuolar t-SNARE, Vam3p (but not CPS and CPY), while deletion of AP-1 and AP-2 subunits has no effect on vacuolar protein delivery. This study, therefore, provides evidence that the AP-3 complex functions in cargo-selective protein transport from the Golgi to the vacuole/lysosome.

Phosphoinositide signaling and the regulation of membrane trafficking in yeast

Phosphoinositides are key regulators of diverse cellular processes in eukaryotic cells. Genetic studies in yeast have advanced our understanding of how phosphoinositide-signaling pathways regulate membrane trafficking. Enzymes required for the synthesis (kinases) and turnover (phosphatases) of distinct phosphoinositides have been identified and several downstream effector molecules linked to phosphoinositide signaling have recently been characterized.

The AP-3 complex: a coat of many colours

A new adaptor protein complex, termed AP-3, has recently been identified in mammalian cells, and genetic studies in yeast have revealed a functional role for the AP-3 complex in cargo-selective transport via a new alternative trafficking pathway from the Golgi to the vacuole/lysosome. Here, the authors review what is currently known about the AP-3 complex and discuss recent insight into its function in multicellular organisms that has come from the finding that mutations in AP-3 subunits correspond to classical mutations in Drosophila and mice.

Bro1 is an endosome-associated protein that functions in the MVB pathway in Saccharomyces cerevisiae

Multivesicular bodies are late endosomal compartments containing lumenal vesicles that are formed by inward budding of the limiting endosomal membrane. In the yeast Saccharomyces cerevisiae, integral membrane proteins are sorted into the lumenal vesicles of multivesicular bodies, and this process requires the class E subset of VPS genes. We show that one of the class E VPS genes, BRO1/VPS31, encodes a cytoplasmic protein that associates with endosomal compartments. The dissociation of Bro1 from endosomes requires another class E Vps protein, Vps4, which is an ATPase that also regulates the endosomal dissociation of ESCRT-III, a complex of four class E Vps proteins (Vps2, Vps20, Vps24 and Snf7/Vps32) that oligomerize at the endosomal membrane. We also show that the endosomal association of Bro1 is specifically dependent on one of the ESCRT-III components, Snf7. Our data suggest that the function of Bro1 in the MVB pathway takes place on endosomal membranes and occurs in concert with or downstream of the function of the ESCRT-III complex.

Bro1 coordinates deubiquitination in the multivesicular body pathway by recruiting Doa4 to endosomes

Ubiquitination directs the sorting of cell surface receptors and other integral membrane proteins into the multivesicular body (MVB) pathway. Cargo proteins are subsequently deubiquitinated before their enclosure within MVB vesicles. In Saccharomyces cerevisiae, Bro1 functions at a late step of MVB sorting and is required for cargo protein deubiquitination. We show that the loss of Bro1 function is suppressed by the overexpression of DOA4, which encodes the ubiquitin thiolesterase required for the removal of ubiquitin from MVB cargoes. Overexpression of DOA4 restores cargo protein deubiquitination and sorting via the MVB pathway and reverses the abnormal endosomal morphology typical of bro1 mutant cells, resulting in the restoration of multivesicular endosomes. We further demonstrate that Doa4 interacts with Bro1 on endosomal membranes and that the recruitment of Doa4 to endosomes requires Bro1. Thus, our results point to a key role for Bro1 in coordinating the timing and location of deubiquitination by Doa4 in the MVB pathway.

The Arabidopsis AAA ATPase SKD1 Is Involved in Multivesicular Endosome Function and Interacts with Its Positive Regulator LYST-INTERACTING PROTEIN5

In yeast and mammals, the AAA ATPase Vps4p/SKD1 (for Vacuolar protein sorting 4/SUPPRESSOR OF K+ TRANSPORT GROWTH DEFECT1) is required for the endosomal sorting of secretory and endocytic cargo. We identified a VPS4/SKD1 homolog in Arabidopsis thaliana, which localizes to the cytoplasm and to multivesicular endosomes. In addition, green fluorescent protein–SKD1 colocalizes on multivesicular bodies with fluorescent fusion protein endosomal Rab GTPases, such as ARA6/RabF1, RHA1/RabF2a, and ARA7/RabF2b, and with the endocytic marker FM4-64. The expression of SKD1E232Q, an ATPase-deficient version of SKD1, induces alterations in the endosomal system of tobacco (Nicotiana tabacum) Bright Yellow 2 cells and ultimately leads to cell death. The inducible expression of SKD1E232Q in Arabidopsis resulted in enlarged endosomes with a reduced number of internal vesicles. In a yeast two-hybrid screen using Arabidopsis SKD1 as bait, we isolated a putative homolog of mammalian LYST-INTERACTING PROTEIN5 (LIP5)/SKD1 BINDING PROTEIN1 and yeast Vta1p (for Vps twenty associated 1 protein). Arabidopsis LIP5 acts as a positive regulator of SKD1 by increasing fourfold to fivefold its in vitro ATPase activity. We isolated a knockout homozygous Arabidopsis mutant line with a T-DNA insertion in LIP5. lip5 plants are viable and show no phenotypic alterations under normal growth conditions, suggesting that basal SKD1 ATPase activity is sufficient for plant development and growth.

The multiple personalities of Alix.

Alix is a cytosolic protein in mammalian cells that was originally identified on the basis of its association with pro-apoptotic signaling. More recent evidence has established that Alix has a hand in regulating other cellular mechanisms, including endocytic membrane trafficking and cell adhesion. Although Alix appears to participate directly in these various activities, the role it plays in each process has largely been inferred from the functions of proteins with which it interacts. For example, recruitment of Alix to endosomes is mediated by its N-terminal Bro1 domain, the structure of which was recently solved for its yeast orthologue, Bro1. The diversity of Alix functions is due to its proline-rich C-terminus, which provides multiple protein-binding sites. With this blueprint in hand, we can now ask whether Alix acts simply as an adaptor that links different proteins into networks or, instead, contributes a specific function to distinct molecular machineries.