Arfaptin 1, a putative cytosolic target protein of ADP-ribosylation factor, is recruited to Golgi membranes.
ADP-ribosylation factors (ARFs) have been implicated in vesicle transport in the Golgi complex. Employing yeast two-hybrid screening of an HL60 cDNA library using a constitutively active mutant of ARF3 (ARF3.Q71L), as a probe, we have identified a cDNA encoding a novel protein with a calculated molecular mass of 38.6 kDa, which we have named arfaptin 1. The mRNA of arfaptin 1 was ubiquitously expressed, and recombinant arfaptin 1 bound preferentially to class I ARFs, especially ARF1, but only in the GTP-bound form. The interactions were independent of myristoylation of ARF. Arfaptin 1 in cytosol was recruited to Golgi membranes by ARF in a guanosine 5'-O-(3-thiotriphosphate)-dependent and brefeldin A-sensitive manner. When expressed in COS cells, arfaptin 1 was localized to the Golgi complex. The yeast two-hybrid system yielded another clone, which encoded a putative protein, which we have named arfaptin 2. This consisted of the same number of amino acids as arfaptin 1 and was 60% identical to it. Arfaptin 2 was also ubiquitously expressed and bound to the GTP-, but not GDP-liganded form of class I ARFs, especially ARF1. These results suggest that arfaptins 1 and 2 may be direct target proteins of class 1 ARFs. Arfaptin 1 may be involved in Golgi function along with ARF1.
Kanoh
H
H
Howard Hughes Medical Institute and the Department of Molecular Physiology and Biophysics and Pharmacology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, USA.
Williger
B T
BT
Exton
J H
JH
D020823:ADP-Ribosylation Factor 1; D020727:ADP-Ribosylation Factors; D048868:Adaptor Proteins, Signal Transducing; D000246:Adenosine Diphosphate Ribose / Q000378:metabolism*; D000262:Adenylyl Cyclases / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D019556:COS Cells; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism*; D018076:DNA, Complementary / Q000737:chemistry; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006056:Golgi Apparatus / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D018922:HL-60 Cells; D006801:Humans; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D011943:Receptors, Adrenergic, beta / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid
C105088:ARFIP1 protein, human; C101158:ARFIP2 protein, human; D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; D018076:DNA, Complementary; D011943:Receptors, Adrenergic, beta; D000246:Adenosine Diphosphate Ribose; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate; D019204:GTP-Binding Proteins; D020823:ADP-Ribosylation Factor 1; D020727:ADP-Ribosylation Factors; D000262:Adenylyl Cyclases