Vectorial proteomics reveal targeting, phosphorylation and specific fragmentation of polymerase I and transcript release factor (PTRF) at the surface of caveolae in human adipocytes.
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Aboulaich N, Vainonen JP, Stralfors P, Vener AV
Vectorial proteomics reveal targeting, phosphorylation and specific fragmentation of polymerase I and transcript release factor (PTRF) at the surface of caveolae in human adipocytes.
Biochem J. 2004 Oct 15;383(Pt 2):237-48.
- PubMed ID
- 15242332 [ View in PubMed]
- Abstract
Caveolae, the specialized invaginations of plasma membranes, formed sealed vesicles with outwards-orientated cytosolic surface after isolation from primary human adipocytes. This morphology allowed differential, vectorial identification of proteins at the opposite membrane surfaces by proteolysis and MS. Extracellular-exposed caveolae-specific proteins CD36 and copper-containing amine oxidase were concealed inside the vesicles and resisted trypsin treatment. The cytosol-orientated caveolins were efficiently digested by trypsin, producing peptides amenable to direct MS sequencing. Isolation of peripheral proteins associated with the cytosolic surface of caveolae revealed a set of proteins that contained nuclear localization signals, leucine-zipper domains and PEST (amino acid sequence enriched in proline, glutamic acid, serine and threonine) domains implicated in regulation by proteolysis. In particular, PTRF (polymerase I and transcript release factor) was found as a major caveolae-associated protein and its co-localization with caveolin was confirmed by immunofluorescence confocal microscopy. PTRF was present at the surface of caveolae in the intact form and in five different truncated forms. Peptides (44 and 45 amino acids long) comprising both the PEST domains were sequenced by nanospray-quadrupole-time-of-flight MS from the full-length PTRF, but were not found in the truncated forms of the protein. Two endogenous cleavage sites corresponding to calpain specificity were identified in PTRF; one of them was in a PEST domain. Both cleavage sites were flanked by mono- or diphosphorylated sequences. The phosphorylation sites were localized to Ser-36, Ser-40, Ser-365 and Ser-366 in PTRF. Caveolae of human adipocytes are proposed to function in targeting, relocation and proteolytic control of PTRF and other PEST-domain-containing signalling proteins.
DrugBank Data that Cites this Article
- Polypeptides
Name UniProt ID Acetyl-CoA acetyltransferase, mitochondrial P24752 Details Aldehyde dehydrogenase, mitochondrial P05091 Details Succinate dehydrogenase [ubiquinone] flavoprotein subunit, mitochondrial P31040 Details Membrane primary amine oxidase Q16853 Details Cytochrome b-c1 complex subunit 1, mitochondrial P31930 Details Guanine nucleotide-binding protein G(s) subunit alpha isoforms short P63092 Details ATP synthase subunit beta, mitochondrial P06576 Details Annexin A5 P08758 Details Citrate synthase, mitochondrial O75390 Details Guanine nucleotide-binding protein G(i) subunit alpha-1 P63096 Details 60 kDa heat shock protein, mitochondrial P10809 Details 3-ketoacyl-CoA thiolase, mitochondrial P42765 Details