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Title: ADP-ribosylation factor-like protein 4C (ARL4C) interacts with galectin-3 during oocyte development and embryogenesis in rainbow trout (Oncorhynchus mykiss)

Author
item ZHANG, XIAOMING - West Virginia University
item Rexroad, Caird
item YAO, JIANBO - West Virginia University

Submitted to: Society for the Study of Reproduction Annual Meeting
Publication Type: Abstract Only
Publication Acceptance Date: 4/9/2010
Publication Date: 7/30/2010
Citation: Zhang, X., Rexroad Iii, C.E., Yao, J. 2010. ADP-ribosylation factor-like protein 4C (ARL4C) interacts with galectin-3 during oocyte development and embryogenesis in rainbow trout (Oncorhynchus mykiss) [abstract]. Society for the Study of Reproduction Annual Meeting. Paper No. 25.

Interpretive Summary:

Technical Abstract: ADP-ribosylation factor-like protein 4 (ARL4) is a GTP-binding protein which belongs to the ADP-ribosylation factor protein (ARF) superfamily of small GTPases. ARL4 has been shown to be mainly related to the development of male germ cells and embryogenesis in mouse. To investigate the role of ARL4 in oocyte and embryonic development in rainbow trout, we cloned the cDNA for rainbow trout ARL4C (RtARL4C), determined its cellular localization, and attempted to identify its interacting proteins in oocytes/embryos. The predicted RtARL4C protein contains 151 amino acids which is highly homologous to other ARL proteins found in mammals and fishes. Biochemical study of purified recombinant RtARL4C protein showed that RtARL4C has GTPase activity without the assistance of GTPase-activating protein. Fluorescence analysis demonstrated that GFP-tagged RtARL4C is primarily localized in the plasma membrane and cytoplasm of RTG cells and predominantly present in the plasma membrane of Hek-293 cells. Analysis of in vivo expression of RtARL4C mutants showed that localization of RtARL4C at the plasma membrane is N-terminal myristoylation dependent. To identify proteins that mediate ARL-dependent signaling in fish development, yeast two-hybrid screening was performed with a rainbow trout oocyte/embryo library. An interaction between RtARL4C and Galectin-3 (RtGal3) was identified. Galectin-3 is a ß-galactoside-binding animal lectin and has pleiotropic biological functions such as cell growth, cell adhesion, cell proliferation, apoptosis and mRNA processing. The interaction between RtARL4C and RtGal3 was validated by GST pull down assay, co-immunoprecipitation and co-localization in mammalian cells and fish cells. The domains necessary for RtGal3 binding were subsequently determined using different RtARL4C mutants. The analysis revealed that interaction of RtARL4C and RtGal3 requires two GTP-binding sites and the N-terminal myristoylation site. The data suggest that RtARL4C may be involved in a signal transduction pathway that regulates membrane trafficking during oocyte development and embryogenesis in rainbow trout.