Cytosolic Arl2 is complexed with cofactor D and protein phosphatase 2A.
Arl2 is a member of the ADP-ribosylation factor family of 20-kDa GTPases that is highly conserved in eukaryotes. Recent results revealed that a portion of cellular Arl2 and its binding partner, BART, localize to mitochondria. Because approximately 90% of cellular Arl2 is cytosolic, we investigated properties of the soluble protein and found that it is stably bound in a complex that migrates in gel filtration medium with a predicted molecular mass of approximately 300 kDa. This complex was purified approximately 500-fold from the soluble fraction of bovine brain. Protein components were identified by mass spectroscopy and revealed the presence of four other proteins that include the tubulin folding cochaperone cofactor D and all three subunits of at least two protein phosphatase 2A (PP2A) protein phosphatase trimers. The presence of more than one PP2A B-type subunit and the low stoichiometry of Arl2 indicate that the purified preparation still contains a mixture of complexes that cannot currently be completely resolved. Thus, although all the soluble Arl2 in bovine brain is in high molecular mass complexes, only a portion of the total cellular cofactor D and PP2A are associated with the Arl2. We further show that the Arl2 in the complex cannot bind GTP and that complexed cofactor D does not efficiently participate in tubulin refolding reactions in a manner comparable with free cofactor D. Our data suggest functional roles for the cytosolic Arl2 complex in modulating tubulin and microtubule behavior as well as a possible role in apoptosis.
Shern
Jack F
JF
Department of Biochemistry, Emory University School of Medicine, Atlanta, Georgia 30322, USA.
Sharer
J Daniel
JD
Pallas
David C
DC
Bartolini
Francesca
F
Cowan
Nicholas J
NJ
Reed
Matthew S
MS
Pohl
Jan
J
Kahn
Richard A
RA
D000818:Animals; D017209:Apoptosis; D001921:Brain / Q000378:metabolism; D002417:Cattle; D002850:Chromatography, Gel; D003600:Cytosol / Q000378:metabolism*; D019204:GTP-Binding Proteins / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D013058:Mass Spectrometry; D008869:Microtubule-Associated Proteins / Q000378:metabolism*; D008870:Microtubules / Q000378:metabolism; D008928:Mitochondria / Q000378:metabolism; D010749:Phosphoprotein Phosphatases / Q000378:metabolism*; D011233:Precipitin Tests; D011485:Protein Binding; D017510:Protein Folding; D054648:Protein Phosphatase 2; D051381:Rats; D013696:Temperature; D013997:Time Factors; D014404:Tubulin / Q000737:chemistry / Q000378:metabolism
D008869:Microtubule-Associated Proteins; C418662:TBCD protein, human; D014404:Tubulin; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D010749:Phosphoprotein Phosphatases; D054648:Protein Phosphatase 2; C485820:Arl2 protein, rat; D019204:GTP-Binding Proteins