RESUMEN
Vesicular integral protein of 36 kDa (VIP36) is an intracellular lectin recognizing high-mannose type glycans and is highly expressed in salivary glands, especially the parotid gland, which secretes alpha-amylase in large quantities. Here immunoelectron microscopy demonstrated that VIP36 was primarily localized to secretory vesicles in the glandula parotis of the rat, where alpha-amylase also resided. A secretory vesicle fraction, prepared by Percoll density gradient centrifugation, contained both VIP36 and alpha-amylase. Moreover, alpha-amylase that was localized to these secretory vesicles contained high-mannose type glycans. In addition, VIP36 coprecipitated with alpha-amylase in an endo H treatment-sensitive manner. These results suggest that VIP36 is involved in the secretion of alpha-amylase in the rat parotid gland.
Asunto(s)
Lectinas de Unión a Manosa/metabolismo , Glándula Parótida/metabolismo , Vesículas Secretoras/metabolismo , alfa-Amilasas/metabolismo , Animales , Masculino , Microscopía Inmunoelectrónica , Glándula Parótida/ultraestructura , Ratas , Ratas WistarRESUMEN
The vesicular integral membrane protein VIP36 belongs to the family of animal lectins and may act as a cargo receptor trafficking certain glycoproteins in the secretory pathway. Immunoelectron microscopy of GH3 cells provided evidence that endogenous VIP36 is localized mainly in 70-100-nm-diameter uncoated transport vesicles between the exit site on the ER and the neighboring cis-Golgi cisterna. The thyrotrophin-releasing hormone (TRH) stimulation and treatment with actin filament-perturbing agents, cytochalasin D or B or latrunculin-B, caused marked aggregation of the VIP36-positive vesicles and the appearance of a VIP36-positive clustering structure located near the cis-Golgi cisterna. The size of this structure, which comprised conspicuous clusters of VIP36, depended on the TRH concentration. Confocal laser scanning microscopy confirmed the electron microscopically demonstrated distribution and redistribution of VIP36 in these cells. Furthermore, VIP36 colocalized with filamentous actin in the paranuclear Golgi area and its vicinity. This is the first study to show the ultrastructural distribution of VIP36 in the early secretory pathway in GH3 cells. It suggests that actin filaments are involved in glycoprotein transport between the ER and cis-Golgi cisterna by using the lectin VIP36.
Asunto(s)
Retículo Endoplásmico/metabolismo , Aparato de Golgi/metabolismo , Lectinas de Unión a Manosa/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Vesículas Transportadoras/metabolismo , Animales , Línea Celular Tumoral , Citocalasina B/metabolismo , Citocalasina D/metabolismo , Proteínas del Citoesqueleto/metabolismo , Retículo Endoplásmico/ultraestructura , Aparato de Golgi/ultraestructura , Microscopía Inmunoelectrónica , Inhibidores de la Síntesis del Ácido Nucleico/metabolismo , Ratas , Hormona Liberadora de Tirotropina/metabolismo , Vesículas Transportadoras/química , Vesículas Transportadoras/ultraestructuraRESUMEN
VIP36 (36-kD vesicular integral membrane protein), originally purified from Madin-Darby canine kidney (MDCK) epithelial cells, belongs to a family of animal lectins and may act as a cargo receptor. To understand its role in secretory processes, we performed morphological analysis of the rat parotid gland. Immunoelectron microscopy provided evidence that endogenous VIP36 is localized in the trans-Golgi network, on immature granules, and on mature secretory granules in acinar cells. Double-staining immunofluorescence experiments confirmed that VIP36 and amylase co-localized in the apical regions of the acinar cells. This is the first study to demonstrate that endogenous VIP36 is involved in the post-Golgi secretory pathway, suggesting that VIP36 plays a role in trafficking and sorting of secretory and/or membrane proteins during granule formation.