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.
Brûlé
Sophie
S
Centre de recherche en reproduction animale, Faculté de médecine vétérinaire, Université de Montréal, St-Hyacinthe, Québec, Canada.
Faure
Robert
R
Doré
Monique
M
Silversides
David W
DW
Lussier
Jacques G
JG
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