Biochem. J. (2004) 383
(237–248) (Printed in Great Britain)
Vectorial proteomics reveal targeting, phosphorylation and specific fragmentation of polymerase I and transcript release factor (PTRF) at the surface of caveolae in human adipocytes
Nabila ABOULAICH, Julia P. VAINONEN, Peter STRÅLFORS1 and Alexander V. VENER1
Division of Cell Biology and Diabetes Research Centre, Faculty of Health Sciences, Linköping University, SE58185 Linköping, Sweden
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.
Key words: caveolae, human adipocyte, MS, PEST sequence, polymerase I and transcript release factor (PTRF), proteolysis.
Abbreviations used: CID, collision-induced dissociation; EHD, Eps15 homology domain; EHD2, EHD-containing protein 2; ESI, electrospray ionization; IMAC, immobilized metal affinity chromatography; MALDI–TOF-MS, matrix-assisted laser-desorption ionization–time-of-flight mass spectrometry; PTRF, polymerase I and transcript release factor; SDPR, serum deprivation response gene product; SRBC, serum deprivation response-related gene product that binds to C-kinase.
1Correspondence may be addressed to either author (email aleve@ibk.liu.se and peter.stralfors@ibk.liu.se).
Received 19 April 2004/2 July 2004; accepted 9 July 2004
Published as BJ Immediate Publication 9 July 2004, DOI 10.1042/BJ20040647
The Biochemical Society, London ©2004
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