Vigilin is co-localized with 80S ribosomes and binds to the ribosomal complex through its C-terminal domain.
The biological relevance of vigilin a ubiquitous multi (KH)-domain protein is still barely understood. Investigations over the last years, however, provided evidence for a possible involvement of vigilin in the nucleo-cytoplasmic transport of tRNA and in the subsequent association of tRNA with ribosomes. We therefore investigated the potential association of vigilin with 80S ribosomes. Immunostaining, gel filtration, westernblot analysis of polyribosomes and high salt treatment of 80S ribosomes isolated from fresh human placenta were applied to analyze the possible association of vigilin with ribosomes. Overlay assays were performed to examine whether vigilin is capable of binding to ribosomal proteins. Immunostaining of HEp-2 cells, gel filtration of a cytoplasmic extract of HEp-2 cells and westernblot analysis of isolated 80S ribosomes clearly demonstrate that vigilin is bond to the ribosomal complex. Vigilin detaches from the ribosomal complex under the influence of high salt concentrations. We present data that radioactively labeled human vigilin interacts directly with a subset of ribosomal proteins from both subunits. We were able to narrow down the putative binding region to the C-terminal domain by using vigilin mutant constructs. Therefore our results provide strong evidence that vigilin is bond to the ribosomal complex and underline the hypothesis that vigilin might be involved in the link between tRNA-export and the channeled tRNA-cycle on ribosomes.
Vollbrandt
Tillman
T
Department of Medical Molecular Biology, University of Lübeck, Ratzeburger Allee 160, Lübeck D-23538, Germany.
Willkomm
Dagmar
D
Stossberg
Helge
H
Kruse
Charli
C
D021581:Active Transport, Cell Nucleus; D002352:Carrier Proteins / Q000032:analysis* / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D002466:Cell Nucleolus / Q000648:ultrastructure; D005260:Female; D015870:Gene Expression; D006801:Humans; D010920:Placenta / Q000378:metabolism; D011132:Polyribosomes / Q000378:metabolism*; D011485:Protein Binding; D025941:Protein Interaction Mapping; D017434:Protein Structure, Tertiary; D016601:RNA-Binding Proteins / Q000032:analysis* / Q000235:genetics / Q000378:metabolism*; D012269:Ribosomal Proteins / Q000378:metabolism*; D012270:Ribosomes / Q000737:chemistry; D017384:Sequence Deletion / Q000235:genetics
D002352:Carrier Proteins; D016601:RNA-Binding Proteins; D012269:Ribosomal Proteins; C075197:high density lipoprotein binding protein