Identification and Characterization of ARL4A putative effector, GCC185
Date Issued
2008
Date
2008
Author(s)
Tsai, Yueh-Tso
Abstract
ARL4A is a 22 kDa GTPase which showed to be abundant in testes of pubertal and adult rodents. In addition, previous study also showed that the appearance of mouse ARL4A mRNA during embryonic development coincided temporally with the sequential formation of somites and the establishment of brain compartmentation (Lin et al., 2000). To identify potential ARL4A effectors, Yeast Two Hybrid Screening was conducted. I used pBTM116-ARL4A-Q79L as a bait in a yeast-two hybrid screen of human fetal brain cDNA library. putative effector of ARL4A, golgin GCC185, was chosen for further study. Yeast two-hybrid analysis showed that GCC185 (a.a. 500-733) interacted with wild type ARL4A and ARL4A-Q79L, but not with ARL4A-T34N. Pull down assay demonstrated preferential binding of GCC185 (a.a. 500-733) to MBP-ARL4A-Q79L-His. Polyclonal antisera against GCC185 were raised, and the data of immunostaining using the antisera suggested the antisera can recognize endogenous GCC185. Putative endogenous GCC185 signal recognized by the antisera was supported by the data that this signal colocalized with trans Golgi marker p230/Golgin245. By immunostaining of COS cells depleted of GCC185, a diminish of the TGN signal can be observed using the antiserum, further supporting that the antiserum can recognize endogenous GCC185. CC185 was known to be involved in retrograde transport of Shiga Toxin subunit B (STxB) and mannose 6-phosphate receptors (MPRs). To investigate biological relevance of the interaction between ARL4A and GCC185, we attempted to evaluate whether mutant ARL4A could affect the retrograde transport of STxB and MPRs. I purified STxB and conjugated it with fluorescent dye Cy3 in order to study STxB internalization. Preliminary data did not suggest overexpression of ARL4A mutant could affect the STxB internalization and retrograde transport of MPRs. The experiments on RNAi gene silencing of ARL4A will provide a more definite conclusion.
Subjects
GCC185
GTPase
ARL4A
shiga toxin
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