Rab4 Orchestrates a Small GTPase Cascade for Recruitment of Adaptor Proteins to Early Endosomes

• Published in: Current biology Vol. 24; no. 11; pp. 1187 - 1198
• Main Authors: D’Souza, Ryan S., Semus, Rachel, Billings, Emily A., Meyer, Corey B., Conger, Kathryn, Casanova, James E.
• Format: Journal Article
• Language: English
• Published: England Elsevier Inc 02.06.2014
• Subjects: ADP-Ribosylation Factors - genetics; ADP-Ribosylation Factors - metabolism; Animals; Cercopithecus aethiops; COS Cells; Endosomes - metabolism; GTP Phosphohydrolases - metabolism; Humans; Intracellular Membranes - metabolism; Membrane Proteins - genetics; Membrane Proteins - metabolism; rab4 GTP-Binding Proteins - genetics; rab4 GTP-Binding Proteins - metabolism; Signal Transduction
• ISSN: 0960-9822; 1879-0445
• DOI: 10.1016/j.cub.2014.04.003

Early, sorting endosomes are a major crossroad of membrane traffic, at the intersection of the endocytic and exocytic pathways. The sorting of endosomal cargo for delivery to different subcellular destinations is mediated by a number of distinct coat protein complexes, including adaptor protein 1 (AP-1), AP-3, and Golgi-localized, gamma adaptin ear-containing, Arf-binding (GGAs) protein. Ultrastructural studies suggest that these coats assemble onto tubular subdomains of the endosomal membrane, but the mechanisms of coat recruitment and assembly at this site remain poorly understood. Here we report that the endosomal Rab protein Rab4 orchestrates a GTPase cascade that results in the sequential recruitment of the ADP-ribosylation factor (Arf)-like protein Arl1; the Arf-specific guanine nucleotide exchange factors BIG1 and BIG2; and the class I Arfs, Arf1 and Arf3. Knockdown of Arf1, or inhibition of BIG1 and BIG2 activity with brefeldin A results in the loss of AP-1, AP-3, and GGA-3, but not Arl1, from endosomal membranes and the formation of elongated tubules. In contrast, depletion of Arl1 randomizes the distribution of Rab4 on endosomal membranes, inhibits the formation of tubular subdomains, and blocks recruitment of BIG1 and BIG2, Arfs, and adaptor protein complexes to the endosome. Together these findings indicate that Arl1 links Rab4-dependent formation of endosomal sorting domains with downstream assembly of adaptor protein complexes that constitute the endosomal sorting machinery. •Rab4 marks an early endosomal subdomain for localized adaptor protein recruitment•Rab4 recruits Arl1, which recruits the Arf GEFs BIG1 and BIG2 to early endosomes•BIG1 and BIG2 locally activate class I Arfs, promoting adaptor recruitment to endosomes•Arf1 and Arf3 have nonredundant functions in adaptor protein recruitment How carrier vesicles form from endosomes is poorly understood. D’Souza et al. report that the GTPase Rab4 orchestrates a signaling cascade leading to sequential recruitment of a second GTPase, Arl1; the Arf GEFs BIG1 and BIG2; and two more GTPases, Arf1 and Arf3. The Arfs then nucleate assembly of multiple classes of clathrin adaptors onto early endosomes.