Rab5 regulates endocytic membrane traffic by specifically recruiting cytosolic effector proteins to their site of action on early endosomal membranes. We have characterized a new Rab5 effector complex involved in endosomal fusion events. This complex includes a novel protein, Rabenosyn-5, which, like the previously characterized Rab5 effector early endosome antigen 1 (EEA1), contains an FYVE finger domain and is recruited in a phosphatidylinositol-3-kinase–dependent fashion to early endosomes. Rabenosyn-5 is complexed to the Sec1-like protein hVPS45. hVPS45 does not interact directly with Rab5, therefore Rabenosyn-5 serves as a molecular link between hVPS45 and the Rab5 GTPase. This property suggests that Rabenosyn-5 is a closer mammalian functional homologue of yeast Vac1p than EEA1. Furthermore, although both EEA1 and Rabenosyn-5 are required for early endosomal fusion, only overexpression of Rabenosyn-5 inhibits cathepsin D processing, suggesting that the two proteins play distinct roles in endosomal trafficking. We propose that Rab5-dependent formation of membrane domains enriched in phosphatidylinositol-3-phosphate has evolved as a mechanism for the recruitment of multiple effector proteins to mammalian early endosomes, and that these domains are multifunctional, depending on the differing activities of the effector proteins recruited.

We have stably expressed in HeLa cells a chimeric protein made of the green fluorescent protein (GFP) fused to the transmembrane and cytoplasmic domains of the mannose 6-phosphate/insulin like growth factor II receptor in order to study its dynamics in living cells. At steady state, the bulk of this chimeric protein (GFP-CI-MPR) localizes to the trans-Golgi network (TGN), but significant amounts are also detected in peripheral, tubulo-vesicular structures and early endosomes as well as at the plasma membrane. Time-lapse videomicroscopy shows that the GFP-CI-MPR is ubiquitously detected in tubular elements that detach from the TGN and move toward the cell periphery, sometimes breaking into smaller tubular fragments. The formation of the TGN-derived tubules is temperature dependent, requires the presence of intact microtubule and actin networks, and is regulated by the ARF-1 GTPase. The TGN-derived tubules fuse with peripheral, tubulo-vesicular structures also containing the GFP-CI-MPR. These structures are highly dynamic, fusing with each other as well as with early endosomes. Time-lapse videomicroscopy performed on HeLa cells coexpressing the CFP-CI-MPR and the AP-1 complex whose ␥-subunit was fused to YFP shows that AP-1 is present not only on the TGN and peripheral CFP-CI-MPR containing structures but also on TGN-derived tubules containing the CFP-CI-MPR. The data support the notion that tubular elements can mediate MPR transport from the TGN to a peripheral, tubulo-vesicular network dynamically connected with the endocytic pathway and that the AP-1 coat may facilitate MPR sorting in the TGN and endosomes. INTRODUCTIONThe mannose 6-phosphate receptors (MPRs) are essential components for lysosome biogenesis and cellular homeostasis (Kornfeld, 1992;Ludwig et al., 1995). The primary function of the cation-independent and the cation-dependent mannose 6-phosphate receptors (CI-MPR and CD-MPR) is to sort newly synthesized lysosomal enzymes from the secretory pathway for subsequent transport to endosomal/lysosomal compartments. To carry out their function, the MPRs must bind the common mannose 6-phosphate recognition marker on soluble lysosomal enzymes in the trans-Golgi network (TGN), the last sorting station of the secretory pathway, and be packaged into TGN-derived transport intermediates. After budding, these transport intermediates fuse with endosomes where the MPRs unload their bound ligands. Although the lysosomal enzymes are transported to lysosomes, the MPRs are retrieved to the TGN or occasionally to the plasma membrane. Although the precise pathways followed by the MPRs at the exit of the TGN remain to be better defined, it has become clear during the past years that the sorting of MPRs from the compartments they visit, i.e., TGN, endosomes and plasma membrane, is directed by Article published online ahead of print. Mol. Biol. Cell 10.1091/ mbc.E02-06 -0338. Article and publication date are at www. molbiolcell.org/cgi/doi/10.1091/mbc.E02-06 -0338.□ V Online version of this article contains video material. ...