Cloning and characterization of Vear, a novel Golgi-associated protein involved in vesicle trafficking
|Organizations:||University of Oulu, Faculty of Medicine, Department of Pathology
|Online Access:||PDF Full Text (PDF, 1.2 MB)|
|Persistent link:|| http://urn.fi/urn:isbn:9514264290
|Publish Date:|| 2001-06-20
|Thesis type:||Doctoral Dissertation
|Defence Note:||Academic Dissertation to be presented with the assent of the Faculty of Medicine, University of Oulu, for public discussion in the Auditorium of Pharmacology, on August 24th, 2001, at 12 noon.
Docent Esa Kuismanen
Professor Lauri Pelliniemi
The control and maintenance of the character, number and protein, carbohydrates and lipid composition of intracellular compartments in a changing environment is one of the fundamental features of a living cell. It is effected, to a large measure, by vesicular traffic which connects the various cellular compartments and handles the transportation of cargo between them. Movement of cargo occurs through a transport system in membrane-bounded containers called vesicles. Vesicles originate at the donor membrane from which they are transported to target organelles where they fuse with the acceptor membrane and deliver their cargo. At the donor site, cytosolic coat proteins or 'coats' bind to the donor membrane together with GTP (guanosine 5'-triphosphate)-binding regulatory proteins first to deform a bud, which is then pinched off as a coated vesicle. During budding and targeting events, a number of regulatory proteins interact with the coat components. Currently, several different coat proteins and their adaptor proteins are known.
The purpose of this study was to characterize novel components participitating in intracellular vesicle transport. By using computer analysis and EST (expressed sequence tag) database searches, a previously unknown protein was found. Sequencing revealed the presence of a novel protein of 613 amino acids with a calculated molecular mass of 67,149 Da. Based on its structural features, possessing both a VHS domain and an "ear" domain, we named the protein Vear.
With its VHS domain in its NH2 terminus, Vear shows similarity to several endocytosis-associated proteins. With the "ear" domain in its C-terminus, it resembles γ-adaptin, a heavy subunit of the AP-1 complex. Vear mRNA showed a widespread distribution in tissues, with high amounts of mRNA in the kidney, skeletal muscle, and cardiac muscle. At the subcellular level, Vear was localized to the Golgi complex in which it colocalized with the trans-Golgi marker γ-adaptin. The preferential membrane-association was demonstrated by subcellular fractionation in which Vear partitioned with the total membrane fraction. Golgi-associated subcellular localization for Vear was sensitive to a treatment with the fungal metabolite brefeldin A, suggesting an ARF (ADP-ribosylation factor)-dependent recruitment onto membranes. In transfection studies, the full-length Vear assembled on and caused structural "compaction" of the Golgi complex, while overexpression of the "ear" domain alone showed diffuse Golgi-localization without "compaction". The VHS domain, on the other hand, was mainly vesicle- and plasma membrane associated and did not show any association with Golgi. In skeletal muscle, Vear was detected preferentially in type I cells by immunohistochemistry and immunofluorescence microscopy. In normal kidney, Vear was exclusively present in glomerular epithelial cells (podocytes) and Vear protein was expressed in a developmentally regulated manner during glomerulogenesis. By immunolabeling electron microscopy, Vear was seen in vesicular and membrane structures adjacent to the Golgi complex. Vear was also abundant in the gastrointestinal tract in cells active in secretion.
The results indicate that Vear is a novel vesicle transport-associated protein, detected mainly in the Golgi complex and localized in tissues in a highly cell-type specific manner.
Acta Universitatis Ouluensis. D, Medica
|Copyright information:||This publication is copyrighted. You may download, display and print it for your own personal use. Commercial use is prohibited.|