title: Identification of the P-body component PATL1 as a novel ALG-2-interacting protein by in silico and far-Western screening of proline-rich proteins.
abstract: ALG-2 (also named PDCD6) is a 22-kDa Ca(2+)-binding protein that belongs to the penta-EF-hand family including calpain small subunit and interacts with various proteins such as ALIX and Sec31A at their specific sites containing an ALG-2-binding motif (ABM) present in their respective Pro-rich region (PRR). In this study, to search for novel ALG-2-interacting proteins, we first performed in silico screening of ABM-containing PRRs in a human protein database. After selecting 17 sequences, we expressed the PRR or full-length proteins fused with green fluorescent protein (GFP) in HEK293T cells and analysed their abilities to bind to ALG-2 by Far-Western blotting using biotinylated ALG-2 as a probe. As a result, we found 10 positive new ALG-2-binding candidates with different degrees of binding ability. For further investigation, we selected PATL1 (alternatively designated Pat1b), a component of the P-body, which is a cytoplasmic non-membranous granule composed of translation-inactive mRNAs and proteins involved in mRNA decay. Interactions between endogenous PATL1 and ALG-2 proteins were demonstrated by a co-immunoprecipitation assay using their specific antibodies. Furthermore, in immunofluorescence microscopic analyses, PATL1 as well as DCP1A, a well-known P-body marker, co-localized with a subset of ALG-2. This is the first report showing interaction of ALG-2 with a P-body component.
authors.affiliation: Department of Applied Molecular Biosciences, Graduate School of Bioagricultural Sciences, Nagoya University, Chikusa-ku, Nagoya, Japan.
authors.forename: Kanae
authors.initials: K
authors.lastname: Osugi
authors.forename: Hironori
authors.initials: H
authors.lastname: Suzuki
authors.forename: Tomomi
authors.initials: T
authors.lastname: Nomura
authors.forename: Yasuo
authors.initials: Y
authors.lastname: Ariumi
authors.forename: Hideki
authors.initials: H
authors.lastname: Shibata
authors.forename: Masatoshi
authors.initials: M
authors.lastname: Maki
mesh_terms: D051017:Apoptosis Regulatory Proteins / Q000378:metabolism*; D026481:Blotting, Far-Western; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D002478:Cells, Cultured; D019295:Computational Biology; D004268:DNA-Binding Proteins / Q000378:metabolism*; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D011392:Proline / Q000032:analysis*; D011485:Protein Binding
chemical_list: D051017:Apoptosis Regulatory Proteins; D002135:Calcium-Binding Proteins; D004268:DNA-Binding Proteins; C526510:PATL1 protein, human; C097753:PDCD6 protein, human; D011392:Proline