Abstract
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.
Highlights
ADP-ribosylation factors (ARFs),1 which were originally identified and purified by their ability to enhance the ADPribosyltransferase activity of cholera toxin, comprise a distinct subfamily of Ras-related small GTP-binding proteins and have been found in all eukaryotic cells from yeast to human [1]
We have identified two novel proteins, termed arfaptins2 1 and 2, which interact with class I ARFs only in their GTP-bound conformation
Identification of an ARF3-interacting Protein by Yeast Twohybrid Screening—To identify molecules that act as downstream effectors of ARF, we employed the yeast two-hybrid system and screened an HL60 cDNA library using a constitutively active mutant of ARF3 (ARF31⁄7Q71L) [16, 29] as a target
Summary
ADP-ribosylation factors (ARFs), which were originally identified and purified by their ability to enhance the ADPribosyltransferase activity of cholera toxin, comprise a distinct subfamily of Ras-related small GTP-binding proteins and have been found in all eukaryotic cells from yeast to human [1]. Like other members of the Ras superfamily, ARF proteins transmit signals to downstream effectors in a cyclical and guanine nucleotide-dependent manner Conformational differences between their GDP- and GTP-bound forms determine their interaction with regulatory proteins, namely a guanine nucleotide exchange protein (GEP) and a GTPase-activating protein (GAP). A form of mammalian phospholipase D has been cloned, and the recombinant enzyme expressed in Sf9 cells has been shown to be activated by recombinant ARF1 [15]. This does not prove direct interaction between ARF and phospholipase D, it strongly suggests that it is a downstream effector. Arfaptin 1 was shown to be recruited to Golgi membranes by GTP-bound ARF
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