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:NH2 (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.