| Summary: | 碩士 === 國立成功大學 === 生命科學系 === 104 === ADP-ribosylation factors (Arfs) are a family of 20-kDa guanine nucleotide-binding proteins which are involved in regulation of several pathways of intracellular trafficking.
Similar with other small GTPase, activation of Arfs is catalyzed by the Sec7 domain of Arf guanine nucleotide exchange factors (GEFs), which can catalyze the replacement of Arf-bound GDP by GTP. Two brefeldin A (BFA)-inhibited guanine nucleotide-exchange proteins, BIG1 (~200-kDa) and BIG2 (~190-kDa), were purified together in 〉670-kDa
multiprotein complexes from bovine brain cytosol. BIG1 and BIG2 were 90% identical in the ~200-amino-acid Sec7 domains. Besides the catalytic GEF activity of the Sec7 domain,
there are additional highly conserved sequences in the N and C termini of BIG1 and BIG2 that remain less defined. Here, I used the yeast two-hybrid system which BIG1NSec7 (a.a.
1-885, contained Sec7 domain) and BIG1C (a.a. 886-1849) as bait to screen interaction with a human fetal brain cDNA library respectively. We identified several candidate
proteins which related to microtubule assembly, including microtubule-associated protein 1B-light chain 1 (MAP1B-LC1, a.a. 2262-2468) and α-tubulin. Interactive regions of BIG1
to MAP1B-LC1 and α-tubulin were narrowed down by yeast two-hybrid assay. Sec7 domain (a.a. 698-885) of BIG1 is responsible for interaction with MAP1B-LC1, and α-tubulin interacted with the N-terminal structures, but not Sec7 domain of BIG1. Alpha-tubulin is well known as a major component of microtubules. Function of MAP1B are reported to regulate the polymerization of microtubule and actin microfilaments, and involves in axonal elongation, neuronal migration, and axonal guidance. These newly recognized interactions should let us understand better the mechanisms through which BIG1 may integrate local events in membrane trafficking with longer-range transport processes.
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