Identification and Characterization of ARL4D Interacting Protein VPS4A and Establishment and Phenotypic Function Study of ARL4D Knockout Mice
Date Issued
2009
Date
2009
Author(s)
Chen, Wu-Shan
Abstract
ARL 4D is a regulated developmentally protein which belongs to ADP-ribosylation factor (ARF) small GTP binding protein family. In previous researches, our lab observed the heterogeneous distributions of constitutively inactive form of ARL4D (ARL4DT35N) at endosomes, mitochondria, and aggresome-like compartments in COS-7 cells. Indeed, ARL4DT35N overexpression diminished mitochondria membrane potential. To characterize function of ARL4DT35N, yeast two-hybrid screening was performed to identify the interacting partner in the mouse embryonic 7-day library.n interacting protein vacuolar protein sorting factor 4A (VPS4A) was chosen for further study. VPS4A (a.a. 286-437) interacts with ARL4DT35N specifically and directly in a GDP-dependent manner. ARL4DT35N and VPS4A co-overexpression in HeLa cells could enhance the cytokinesis inhibition induced by VPS4A. This increase was similar to dominant-negative VPS4AK173Q overexpression. In addition, ARL4D knockdown by siRNA would reduce cytokinesis inhibition induced by VPS4AK173Q. Together, these data suggested ARL4DT35N might affect the function of VPS4A, and subsequently affect the cytokinesis.RL4D knockout mice have been generated to investigate the physiological function of ARL4D. ARL4D knockout mice were viable and fertile. However, there were many vacuoles in the liver section of male ARL4D knockout mice by hematoxylin and eosin staining, representing primary degeneration of liver function; severe inflammation was also observed in liver of a female ARL4D knockout mouse. Data of blood chemistry analysis indicated the enzyme activities of GOT, GPT, and CPK were higher in ARL4D knockout mice than wild-type and heterozygous mice. This increase was significant in male mice. More samples need to be examined before giving the final conclusion.
Subjects
ARL4D
ARF
yeast twouhybrid screening
cytokinesis
knockout mice
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