Isoform-selective effects of the depletion of ADP-ribosylation factors 1-5 on membrane traffic.
The ADP-ribosylation factors (Arfs) are six proteins within the larger Arf family and Ras superfamily that regulate membrane traffic. Arfs all share numerous biochemical activities and have very similar specific activities. The use of dominant mutants and brefeldin A has been important to the discovery of the cellular functions of Arfs but lack specificity between Arf isoforms. We developed small interference RNA constructs capable of specific depletion of each of the cytoplasmic human Arfs to examine the specificity of Arfs in live cells. No single Arf was required for any step of membrane traffic examined in HeLa cells. However, every combination of the double knockdowns of Arf1, Arf3, Arf4, and Arf5 yielded a distinct pattern of defects in secretory and endocytic traffic, demonstrating clear specificity for Arfs at multiple steps. These results suggest that the cooperation of two Arfs at the same site may be a general feature of Arf signaling and provide candidates at several cellular locations that when paired with data on the localization of the many different Arf guanine nucleotide exchange factors, Arf GTPase activating proteins, and effectors will aid in the description of the mechanisms of specificity in this highly conserved and primordial family of regulatory GTPases.
Volpicelli-Daley
Laura A
LA
Department of Biochemistry, Emory University School of Medicine, Atlanta, GA 30322, USA.
Li
Yawei
Y
Zhang
Chun-Jiang
CJ
Kahn
Richard A
RA
D020727:ADP-Ribosylation Factors / Q000235:genetics / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism*; D020755:Coat Protein Complex I / Q000378:metabolism; D004705:Endocytosis / Q000502:physiology*; D005089:Exocytosis / Q000502:physiology*; D006056:Golgi Apparatus / Q000378:metabolism* / Q000648:ultrastructure; D006367:HeLa Cells; D006801:Humans; D009154:Mutation; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D021381:Protein Transport; D034741:RNA, Small Interfering / Q000235:genetics
D020755:Coat Protein Complex I; D020033:Protein Isoforms; D034741:RNA, Small Interfering; D020727:ADP-Ribosylation Factors