Cyclical action of the WASH complex: FAM21 and capping protein drive WASH recycling, not initial recruitment.
WASH causes actin to polymerize on vesicles involved in retrograde traffic and exocytosis. It is found within a regulatory complex, but the physiological roles of the other four members are unknown. Here we present genetic analysis of the subunits' individual functions in Dictyostelium. Mutants in each subunit are completely blocked in exocytosis. All subunits except FAM21 are required to drive actin assembly on lysosomes. Without actin, lysosomes never recycle vacuolar-type H(+)-adenosine triphosphatase (V-ATPase) or neutralize to form postlysosomes. However, in FAM21 knockout lysosomes, WASH generates excessive, dynamic streams of actin. These successfully remove V-ATPase, neutralize, and form huge postlysosomes. The distinction between WASH and FAM21 phenotypes is conserved in human cells. Thus, FAM21 and WASH act at different steps of a cyclical pathway in which FAM21 mediates recycling of the complex back to acidic lysosomes. Recycling is driven by FAM21's interaction with capping protein, which couples the WASH complex to dynamic actin on vesicles.
CR-UK Beatson Institute for Cancer Research, Glasgow University, Switchback Road, Glasgow G61 1BD, UK.
Laura
L
Park
Peter A
PA
Thomason
Tobias
T
Zech
Jason S
JS
King
Douwe M
DM
Veltman
Michael
M
Carnell
Seiji
S
Ura
Laura M
LM
Machesky
Robert H
RH
Insall
D051344:Actin Capping Proteins / Q000378:metabolism*; D000199:Actins / Q000378:metabolism; D045744:Cell Line, Tumor; D004023:Dictyostelium / Q000235:genetics / Q000378:metabolism*; D005089:Exocytosis; D006801:Humans; D008247:Lysosomes / Q000378:metabolism; D008840:Microfilament Proteins / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D015800:Protozoan Proteins / Q000235:genetics / Q000378:metabolism*; D034622:RNA Interference; D034741:RNA, Small Interfering; D025262:Vacuolar Proton-Translocating ATPases / Q000378:metabolism*; D033921:Vesicular Transport Proteins / Q000235:genetics / Q000378:metabolism*
D051344:Actin Capping Proteins; D000199:Actins; D008840:Microfilament Proteins; D015800:Protozoan Proteins; D034741:RNA, Small Interfering; D033921:Vesicular Transport Proteins; D025262:Vacuolar Proton-Translocating ATPases