Biochemical and Biophysical Research Communications
Afadin- and α-actinin-binding protein ADIP directly binds β′-COP, a subunit of the coatomer complex☆
Section snippets
Materials and methods
Yeast two-hybrid screening. One bait vector, pGBD-mADIP-M (amino acids (aa) 152–436), was constructed by subcloning the insert encoding aa residue of ADIP into pGBD-C1 [8]. Yeast two-hybrid library constructed from the rat lung cDNAs was purchased from Clontech. The two-hybrid screening was performed as described [8]. Standard procedures for yeast manipulations were done as described [9].
Construction of expression vectors. The mammalian expression vectors, pCMV-Flag and pCMV-HA, were designed
Identification of β′-COP as an ADIP-binding protein
To identify an ADIP-binding protein, we performed the yeast two-hybrid screening using the region of ADIP (aa 152–436) containing the three coiled-coil domains as a bait (Fig. 1A). We screened 2 × 105 clones of a rat lung library and obtained 10 positive clones. Three rat clones encoded the carboxyl-terminal portion (aa 370–905) of rat β′-COP (AF002705; GenBank/EMBL/DDBJ) (Fig. 1B). β′-COP contains six internal repeats of the WD40 motif, but the carboxyl-terminal portion (aa 370–905) is not
Discussion
We have shown here that ADIP, an afadin- and α-actinin-binding protein, localizes at the Golgi complex and directly binds β′-COP, a subunit of the coatomer complex. β′-COP is a subunit of the coatomer complex and this complex consists of α-, β-, β′-, γ-, δ-, ε-, and ζ-COPs and is involved in vesicle trafficking from the Golgi to the ER and through the Golgi complex [6], [7]. However, it remains unknown whether the interaction of ADIP with β′-COP is involved in this vesicle trafficking. It has
Acknowledgment
We thank Dr. W. Birchmeier (Max-Delbruck-Center for Molecular Medicine, Berlin, Germany) for providing us with MDCK cells. This investigation was supported by Grants-in-Aid for Scientific Research and for Cancer Research from the Ministry of Education, Science, Sports, Culture, and Technology, Japan (2002, 2003).
References (31)
- et al.
ADIP: a novel afadin- and α-actinin-binding protein localized at cell–cell adherens junctions
J. Biol. Chem.
(2003) Molecular architecture of adherens junctions
Curr. Opin. Cell Biol.
(2001)A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding
Anal. Biochem.
(1976)- et al.
Actin microfilaments are essential for the cytological positioning and morphology of the Golgi complex
Eur. J. Cell Biol.
(1998) - et al.
Budding roles for myosin II on the Golgi
Trends Cell Biol.
(1998) - et al.
Specific isoforms of actin-binding proteins on distinct populations of Golgi-derived vesicles
J. Biol. Chem.
(1999) - et al.
Involvement of LMO7 in the association of two cell–cell adhesion molecules, nectin and E-cadherin, through afadin and α-actinin in epithelial cells
J. Biol. Chem.
(2004) - et al.
Protein trafficking in the exocytic pathway of polarized epithelial cells
Trends Cell Biol.
(2001) - et al.
Nectin and afadin: novel organizers of intercellular junctions
J. Cell Sci.
(2003) - et al.
Nectins and nectin-like molecules: roles in cell adhesion, migration, and polarization
Cancer Sci.
(2003)
The integrin–actin connection, an eternal love affair
EMBO J.
Protein sorting by transport vesicles
Science
Coat proteins and vesicle budding
Science
Genomic libraries and a host strain designed for highly efficient two-hybrid selection in yeast
Genetics
Methods in Yeast Genetics
Cited by (8)
Towards delineation of a developmental α-importome in the mammalian male germline
2013, Biochimica et Biophysica Acta - Molecular Cell ResearchCitation Excerpt :The subcellular distribution of SSX2IP has previously been recorded at cell–cell adherence junctions and in perinuclear regions, such as the Golgi complex [26,30]. In addition, its nuclear localization has been reported in NRK cells [30] and in HeLa cells when the protein included the predicted bipartite NLS-like sequence (285KKEMISLLSPQKKKPR300, referred to as biNLS in this study) [31], which is conserved in the human, mouse and rat orthologues. The cNLS Mapper algorithm identified a putative monopartite NLS between 514 and 523 (RPRQKKPHSV, referred to as pNLS) in rat SSX2IP (Table 2).
SSX2IP: An emerging role in cancer
2007, Biochemical and Biophysical Research CommunicationsCitation Excerpt :1) In mice, investigators have shown that ADIP binds two F-actin-binding proteins, afadin and α-actinin, which suggests it may function in the formation of F-actin bundles and in the linking of the nectin-afadin and cadherin-catenin cell–cell adhesion systems at AJs [8]. Murine ADIP was found to interact with β′-COP, a subunit of the coatomer complex, suggesting a possible role in vesicle trafficking from the Golgi complex to the ER [27]. ( 2) In chickens, LCG was shown to have an expression which was dependent on circadian change and that it interacted with the centrosomal protein γ-tubulin. (
SSX2IP promotes metastasis and chemotherapeutic resistance of hepatocellular carcinoma
2013, Journal of Translational MedicineAnchoring junctions as drug targets: Role in contraceptive development
2008, Pharmacological Reviews
- ☆
Abbreviations: aa, amino acids; ADIP, afadin DIL domain-interacting protein; AJs, adherens junctions; ER, endoplasmic reticulum; F-actin, actin filament; GST, glutathione S-transferase; HA, hemagglutinin; MDCK cell, Madin Darby canine kidney cell; MBP, maltose-binding protein; NRK cell, normal rat kidney cell; pAb, polyclonal antibody; mAb, monoclonal antibody; PAGE, polyacrylamide gel electrophoresis.
- 1
Present address: Department of Geriatric and Respiratory Medicine, Tohoku University School of Medicine, 1-1, Seiryo-machi, Aoba-ku, Sendai 980-8574, Japan.