Immunolocalization of vacuolar system-associated protein-60 (VASAP-60).
We have characterized the localization of the protein termed VASAP-60 in different bovine tissues and cell lines, and have investigated if VASAP-60 interacts with other proteins. Monospecific polyclonal antibodies were raised against distinct fragments of VASAP-60: NH(2) (V(22) to Q(234)), central (A(246) to S(418)), and COOH (L(416) to L(533)). These three antibodies recognized an 88-kDa protein in immunoblotting analysis. The calculated Mr of VASAP-60 derived from its cDNA (60.1 kDa) was significantly lower than its Mr estimated by SDS-PAGE, and this was mainly attributed to the glutamic acid- and aspartic acid-rich composition of its central region (A(246) to S(418)). A 58-kDa proteolytically processed form of VASAP-60 was also identified. Immunocytochemistry demonstrated that VASAP-60 is found predominantly in the perinuclear region, colocalized with calnexin in the endoplasmic reticulum (ER), and partially colocalized with the endocytic marker DAMP. Immunohistochemical localization of VASAP-60 also demonstrated its presence within specialized vesicular structures not related to the ER. Immunoprecipitation using extracts prepared from S(35)Met/Cys metabolically labeled cells demonstrates that VASAP-60 interacts with 116-, 48.5-, and 26.5-kDa proteins. Therefore, VASAP-60 was found to be more widely distributed in the vacuolar system than anticipated, suggesting that VASAP-60 may function in intracellular transport events, rather than being an exclusive component of the quality control mechanism of newly synthesized proteins as thought previously.
Centre de recherche en reproduction animale, Faculté de médecine vétérinaire, Université de Montréal, St-Hyacinthe, Québec, Canada.
Sophie
S
Brûlé
Robert
R
Faure
Monique
M
Doré
David W
DW
Silversides
Jacques G
JG
Lussier
D000818:Animals; D037281:Calnexin / Q000032:analysis / Q000378:metabolism; D002417:Cattle; D002460:Cell Line; D002467:Cell Nucleus / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure; D004136:Dinitrobenzenes / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure; D005455:Fluorescent Antibody Technique; D007202:Indicators and Reagents / Q000378:metabolism; D008565:Membrane Proteins / Q000032:analysis / Q000276:immunology / Q000378:metabolism*; D010446:Peptide Fragments / Q000276:immunology; D011233:Precipitin Tests; D011994:Recombinant Proteins; D014617:Vacuoles / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure
D004136:Dinitrobenzenes; D007202:Indicators and Reagents; D008565:Membrane Proteins; D010446:Peptide Fragments; D011994:Recombinant Proteins; C406303:VASAP-60 protein, Bos taurus; D037281:Calnexin; C042432:3-(2,4-dinitroanilino)-3'-amino-N-methyldipropylamine