Rab4 orchestrates a small GTPase cascade for recruitment of adaptor proteins to early endosomes.
BACKGROUND
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.


RESULTS
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.


CONCLUSIONS
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.
Department of Cell Biology, University of Virginia Health Sciences Centre, P.O. Box 800732, Charlottesville, VA 22908, USA.
Ryan S
RS
D'Souza
Department of Cell Biology, University of Virginia Health Sciences Centre, P.O. Box 800732, Charlottesville, VA 22908, USA.
Rachel
R
Semus
Department of Cell Biology, University of Virginia Health Sciences Centre, P.O. Box 800732, Charlottesville, VA 22908, USA.
Emily A
EA
Billings
Department of Cell Biology, University of Virginia Health Sciences Centre, P.O. Box 800732, Charlottesville, VA 22908, USA.
Corey B
CB
Meyer
Department of Cell Biology, University of Virginia Health Sciences Centre, P.O. Box 800732, Charlottesville, VA 22908, USA.
Kathryn
K
Conger
Department of Cell Biology, University of Virginia Health Sciences Centre, P.O. Box 800732, Charlottesville, VA 22908, USA. Electronic address: jec9e@virginia.edu.
James E
JE
Casanova
D020727:ADP-Ribosylation Factors / Q000235:genetics* / Q000378:metabolism; D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D011992:Endosomes / Q000378:metabolism*; D020558:GTP Phosphohydrolases / Q000378:metabolism; D006801:Humans; D007425:Intracellular Membranes / Q000378:metabolism*; D008565:Membrane Proteins / Q000235:genetics* / Q000378:metabolism; D015398:Signal Transduction; D020695:rab4 GTP-Binding Proteins / Q000235:genetics* / Q000378:metabolism
D008565:Membrane Proteins; C097313:ADP-ribosylation factor related proteins; D020558:GTP Phosphohydrolases; D020727:ADP-Ribosylation Factors; D020695:rab4 GTP-Binding Proteins