Abstract
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.
| Identifer | oai:union.ndltd.org:oulo.fi/oai:oulu.fi:isbn951-42-6429-0 |
| Date | 20 June 2001 |
| Creators | Poussu, A. (Anssi) |
| Publisher | University of Oulu |
| Source Sets | University of Oulu |
| Language | English |
| Detected Language | English |
| Type | info:eu-repo/semantics/doctoralThesis, info:eu-repo/semantics/publishedVersion |
| Format | application/pdf |
| Rights | info:eu-repo/semantics/openAccess, © University of Oulu, 2001 |
| Relation | info:eu-repo/semantics/altIdentifier/pissn/0355-3221, info:eu-repo/semantics/altIdentifier/eissn/1796-2234 |
Page generated in 0.012 seconds