Suzanne R. Pfeffer

Rab GTPases: specifying and deciphering organelle identity and function

Ten years ago, 20 Rab proteins had been identified as organelle-specific GTPases, and two were known to be essential for vesicle targeting in yeast. Today, more than 60 mammalian Rab proteins have been identified. While Rabs were always viewed as key regulatory factors, no one could have anticipated their diversity of functions and multitude of effectors. Rabs organize distinct protein scaffolds within a single organelle and act in a combinatorial manner with their effectors to regulate all stages of membrane traffic.

Rab9 functions in transport between late endosomes and the trans Golgi network.

Rab proteins represent a large family of ras‐like GTPases that regulate distinct vesicular transport events at the level of membrane targeting and/or fusion. We report here the primary sequence, subcellular localization and functional activity of a new member of the rab protein family, rab9. The majority of rab9 appears to be located on the surface of late endosomes. Rab9, purified from Escherichia coli strains expressing this protein, could be prenylated in vitro in the presence of cytosolic proteins and geranylgeranyl diphosphate. In vitro‐prenylated rab9 protein, but not C‐terminally truncated rab9, stimulated the transport of mannose 6‐phosphate receptors from late endosomes to the trans Golgi network in a cell‐free system that reconstitutes this transport step. Rab7, a related rab protein that is also localized to late endosomes, was inactive in the in vitro transport assay, despite its efficient prenylation and capacity to bind and hydrolyze GTP. These results strongly suggest that rab9 functions in the transport of mannose 6‐phosphate receptors between late endosomes and the trans Golgi network. Moreover, our results confirm the observation that a given organelle may bear multiple rab proteins with different biological functions.

Transport-vesicle targeting: tethers before SNAREs.

Protein secretion and the transport of proteins between membrane-bound compartments are mediated by small, membrane-bound vesicles. Here I review what is known about the process by which vesicles are targeted to the correct destination. A growing family of proteins, whose precise modes of action are far from established, is involved in targeting. Despite the wide diversity in the identities of the players, some common themes are emerging that may explain how vesicles can identify their targets and release their cargo at the correct time and place in eukaryotic cells.

Targeting Rab GTPases to distinct membrane compartments.

Rab GTPases are key to membrane-trafficking events in eukaryotic cells, and human cells contain more than 60 Rab proteins that are localized to distinct compartments. The recent determination of the structure of a monoprenylated Rab GTPase bound to GDP-dissociation inhibitor provides new molecular details that are relevant to models of Rab delivery. The further discovery of an integral membrane protein that can dissociate prenylated Rab proteins from GDP-dissociation inhibitor gives new insights into the mechanisms of Rab localization.

TIP47: A Cargo Selection Device for Mannose 6-Phosphate Receptor Trafficking

Mannose 6-phosphate receptors (MPRs) transport newly synthesized lysosomal hydrolases from the Golgi to prelysosomes and then return to the Golgi for another round of transport. We have identified a 47 kDa protein (TIP47) that binds selectively to the cytoplasmic domains of cation-independent and cation-dependent MPRs. TIP47 is present in cytosol and on endosomes and is required for MPR transport from endosomes to the trans-Golgi network in vitro and in vivo. TIP47 recognizes a phenylalanine/tryptophan signal in the tail of the cation-dependent MPR that is essential for its proper sorting within the endosomal pathway. These data suggest that TIP47 binds MPR cytoplasmic domains and facilitates their collection into transport vesicles destined for the Golgi.

RAB GTPASES, DIRECTORS OF VESICLE DOCKING

Rab GTPases represent a large family of Ras-like enzymes that play key roles in the secretory and endocytic pathways. They are located on distinct membrane-bound compartments, and genetic experiments implicate Rabs in the processes by which transport vesicles or membrane-bound compartments recognize their cognate fusion targets (see Refs. 1–4 for review). Because mutant forms of Rab proteins can block protein transport along a given route or actually change the sizes of entire organelles, Rabs obviously play key regulatory roles in membrane trafficking. This minireview will attempt to summarize our current view of what Rabs do. Most Rabs are doubly geranylgeranylated at or near their C termini, which leads to their membrane association. The specificity of Rab localization is provided by structural determinants unique to each family member (5–8) that appear to be recognized by distinct sets of proteins on organelle surfaces (9–12). Like Ras, Rabs cycle between an active, GTPbound form and an inactive, GDP-bound form. Transport vesicles carry Rab proteins with bound GTP; after membrane fusion, GTP hydrolysis converts them into their GDP-bound states. A cytosolic protein, termed GDI, retrieves prenylated, GDP-bound Rab proteins from their fusion targets and recycles them back to their membranes of origin. GDI delivers Rabs to membranes with GDP bound; they are subsequently reactivated by Rab-specific, nucleotide exchange factors (13, 14). At steady state, the bulk of a given Rab is membraneassociated; however, between 10 and 50% can be detected in the cytosol.

Rab GTPases: master regulators of membrane trafficking

Rab GTPases are thought to be likely to catalyze the accurate association of pairs of targeting molecules located on the surfaces of transport vesicles with their corresponding membrane acceptors. Advances during the past year have solidified our understanding of the mechanisms by which Rab proteins are recruited onto nascent transport vesicles and retrieved from their fusion targets. Functional analyses of Rab proteins in living cells have led to the surprising observation that vesicles do not seem to form if the appropriate Rab protein, in its GTP-bound conformation, is not present.

Visualization of Rab9-mediated vesicle transport from endosomes to the trans-Golgi in living cells.

Mannose 6-phosphate receptors (MPRs) are transported from endosomes to the trans-Golgi via a transport process that requires the Rab9 GTPase and the cargo adaptor TIP47. We have generated green fluorescent protein variants of Rab9 and determined their localization in cultured cells. Rab9 is localized primarily in late endosomes and is readily distinguished from the trans-Golgi marker galactosyltransferase. Coexpression of fluorescent Rab9 and Rab7 revealed that these two late endosome Rabs occupy distinct domains within late endosome membranes. Cation-independent mannose 6-phosphate receptors are enriched in the Rab9 domain relative to the Rab7 domain. TIP47 is likely to be present in this domain because it colocalizes with the receptors in fixed cells, and a TIP47 mutant disrupted endosome morphology and sequestered MPRs intracellularly. Rab9 is present on endosomes that display bidirectional microtubule-dependent motility. Rab9-positive transport vesicles fuse with the trans-Golgi network as followed by video microscopy of live cells. These data provide the first indication that Rab9-mediated endosome to trans-Golgi transport can use a vesicle (rather than a tubular) intermediate. Our data suggest that Rab9 remains vesicle associated until docking with the Golgi complex and is rapidly removed concomitant with or just after membrane fusion.

GTP-binding proteins in intracellular transport

One of the most exciting recent discoveries in the area of intracellular protein transport is the finding that many organelles involved in exocytic and endocytic membrane traffic have one or more Ras-like GTP-binding proteins on their cytoplasmic face that are specific for each membranous compartment. These proteins are attractive candidates for regulators of transport vesicle formation and the accurate delivery of transport vesicles to their correct targets.

Yip3 catalyses the dissociation of endosomal Rab-GDI complexes

Human cells contain more than 60 small G proteins of the Rab family, which are localized to the surfaces of distinct membrane compartments and regulate transport vesicle formation, motility, docking and fusion. Prenylated Rabs also occur in the cytosol bound to GDI (guanine nucleotide dissociation inhibitor), which binds to Rabs in their inactive state. Prenyl Rab–GDI complexes contain all of the information necessary to direct Rab delivery onto distinct membrane compartments. The late endosomal, prenyl Rab9 binds GDI with very high affinity, which led us to propose that there might be a ‘GDI-displacement factor’ to catalyse dissociation of Rab–GDI complexes and to enable transfer of Rabs from GDI onto membranes. Indeed, we have previously shown that endosomal membranes contain a proteinaceous factor that can act in this manner. Here we show that the integral membrane protein, Yip3, acts catalytically to dissociate complexes of endosomal Rabs bound to GDI, and to deliver them onto membranes. We propose that the conserved Yip proteins serve as GDI-displacement factors for the targeting of Rab GTPases in eukaryotic cells.