Common and distinct roles for the binding partners Rabenosyn-5 and Vps45 in the regulation of endocytic trafficking in mammalian cells.
In several invertebrate organisms, the Sec1p/Munc18-like protein Vps45 interacts with the divalent Rab4/Rab5 effector, Rabenosyn-5 and carries out multiple functions in the endocytic/secretory pathways. In mammalian cells, Vps45 and Rabenosyn-5 also interact, but the molecular characterization of this binding, and the functional relationship between these two proteins has not been well defined. Here we identify a novel sequence within Rabenosyn-5 required for its interaction with Vps45. We demonstrate that hVps45-depletion decreases expression of Rabenosyn-5, likely resulting from Rabenosyn-5 degradation through the proteasomal pathway. Furthermore, we demonstrate that similar to Rabenosyn-5-depletion, hVps45-depletion causes impaired recycling of beta1 integrins, and a subsequent delay in human fibroblast cell migration on fibronectin-coated plates. Moreover, beta1 integrin recycling could be rescued by reintroduction of siRNA-resistant wild-type Rabenosyn-5, but not a mutant deficient in Vps45 binding. However, unlike Rabenosyn-5-depletion, which induces Golgi fragmentation and decreased recruitment of sorting nexin retromer subunits to the Golgi, hVps45-depletion induces Golgi condensation and accumulation of retromer subunits in the vicinity of the Golgi. In part, these phenomena could be attributed to reduced Syntaxin16 expression and altered localization of both Syntaxin16 and Syntaxin6 upon Vps45-depletion. Overall, these findings implicate hVps45 and Rabenosyn-5 in post early endosome transport, and we propose that their interaction serves as a nexus to promote bidirectional transport along the endosome-to-recycling compartment and endosome-to-Golgi axes.
Rahajeng
Juliati
J
Department of Biochemistry and Molecular Biology and the Eppley Cancer Center, University of Nebraska Medical Center, Omaha, NE 68198-5870, USA.
Caplan
Steve
S
Naslavsky
Naava
N
D000595:Amino Acid Sequence; D000818:Animals; D001692:Biological Transport / Q000502:physiology; D002465:Cell Movement / Q000502:physiology; D002478:Cells, Cultured; D004705:Endocytosis / Q000502:physiology*; D011992:Endosomes / Q000378:metabolism*; D005347:Fibroblasts / Q000166:cytology / Q000502:physiology; D006056:Golgi Apparatus / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D019012:Integrin beta1 / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D011485:Protein Binding; D050765:Qa-SNARE Proteins / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D051438:Syntaxin 16 / Q000378:metabolism; D033921:Vesicular Transport Proteins / Q000235:genetics / Q000378:metabolism*
D019012:Integrin beta1; D050765:Qa-SNARE Proteins; C416362:RBSN protein, human; D034741:RNA, Small Interfering; D051438:Syntaxin 16; C498421:VPS45 protein, human; D033921:Vesicular Transport Proteins; D046988:Proteasome Endopeptidase Complex