Vectorial proteomics reveal targeting, phosphorylation and specific fragmentation of polymerase I and transcript release factor (PTRF) at the surface of caveolae in human adipocytes - PubMed (original) (raw)

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 et al. Biochem J. 2004.

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.

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Figures

Figure 1

Figure 1. Characterization of caveolae purity

(A) Protein patterns in plasma-membrane (PM), caveolae (Cav) and mitochondrial/nuclear (Mit/Nucl) fractions revealed by SDS/PAGE and silver staining (5 μg of protein was loaded in each lane). Molecular-mass standards (kDa) are indicated. The protein band numbers on the right correspond to the numbers of identified proteins in Table 1. (B) Immunoblot analyses of the same cellular fractions (5 μg of protein) with antibodies against caveolin-1 (caveolin) or insulin receptor β-subunit (IR).

Figure 2

Figure 2. Proteolytic treatment of caveolae vesicles

(A) Protein pattern in isolated caveolae before and after treatment with trypsin, as indicated by − and + respectively (Coomassie Blue-stained gel; 8 μg of protein loaded). Positions of molecular-mass standards (kDa) are indicated. Two proteins that resisted tryptic treatment in caveolae are marked with their apparent molecular masses 100 and 70 kDa. (B) Immunoblot analysis of caveolae membrane proteins (3 μg of protein loaded) with antibodies against phosphotyrosine before and after treatment of the vesicles with trypsin, as indicated by − and + respectively. Caveolae were isolated from adipocytes that were stimulated with or without insulin, as indicated. Positions of insulin receptor (IR) and insulin receptor substrate 1 (IRS-1) are shown.

Figure 3

Figure 3. Analyses of caveolins exposed to the outer surface of caveolae vesicles

(A) Immunoblot analysis of caveolae proteins (3 μg of total protein loaded) with anti-caveolin-1 antibody before and after treatment of caveolae with trypsin, as indicated. (B) Full-scan ESI mass spectrum of the peptides released from the surface of caveolae vesicles by trypsin. (C) The product-ion spectrum of the doubly charged peptide ion with m/z 470.2 (also indicated in B) obtained by ESI and CID. The peptide fragment ions are represented as y (C-terminal) or b (N-terminal) fragments in the spectrum and are indicated in the shown peptide sequence obtained from the spectral data and corresponding to the sequence of caveolin-1.

Figure 4

Figure 4. Identification of caveolae-associated proteins

Caveolae vesicles isolated at mild ionic strength and pH conditions were stripped of their associated proteins with 0.5 M carbonate buffer (pH 11). Aliquots containing 5 μg of caveolae proteins before treatment with the carbonate buffer (Tot), the remaining membrane proteins (Memb) and the released proteins (Assoc) were subjected to SDS/PAGE and silver staining. Positions of molecular-mass standards (kDa) are indicated. The numbers on the right correspond to the bands of caveolae-associated proteins that have been identified and described in the same order as in Table 3. CCAO, copper-containing amine oxidase.

Figure 5

Figure 5. Structural similarities between five caveolae-associated proteins

Schematic representation of the domain structure of PTRF, SDPR, SRBC, EHD2 and R-Ras. Polypeptide modules are indicated as follows: LZ, leucine-zipper domain; PEST, PEST sequence; NLS, nuclear localization signal; EH, EHD; EF, calcium-binding motif. Numbers correspond to the first and last amino acids of the particular domain in the protein sequence. Solid and open arrows indicate non-tryptic cleavage sites and phosphorylated serine residues respectively in PTRF.

Figure 6

Figure 6. Distribution of PTRF in subcellular fractions

(A) Immunoblot analysis of mitochondrial/nuclear (Mit/Nucl), cytosol (Cyt), microsome (Mic), plasma-membrane (PM), caveolae (Cav) and caveolae-associated (Assoc) proteins (3 μg of protein loaded in each lane) with antibodies against PTRF. Positions of molecular-mass standards (kDa) are shown. Positions of PTRF fragments are indicated with arrows. (B) Immunoblot analysis of PM, Cav and Nucl proteins (2 μg of protein loaded) with antibodies against the nuclear membrane protein, nucleoporin-p62 (p62). (C) Immunoblot analysis of the same cellular fractions with antibodies against PTRF.

Figure 7

Figure 7. Co-localization of PTRF and caveolin in the plasma membrane of human adipocytes examined by immunofluorescence microscopy

The cytosolic surface of the plasma membrane was labelled with fluorescent antibodies against (A) PTRF (red) and (B) caveolin (green). (C) The superimposed images in (A) and (B) turn yellow where the red and green antibodies are co-localized.

Figure 8

Figure 8. The ESI–CID product-ion spectrum revealing N-terminal acetylation and the PEST sequence of PTRF

The selected quadruply charged peptide ion with m/z 1178.5 (indicated) was subjected to CID. The fragment y (C-terminal) and b (N-terminal) ions having charge state higher than +1 are marked with the corresponding superscript numbers. The sequence of the acetylated 45-amino-acid-long peptide is shown with the numbered fragment ions that were identified in the spectrum.

Figure 9

Figure 9. The ESI–CID product-ion spectra revealing non-tryptic cleavage sites and multiple phosphorylation of PTRF

The detected y (C-terminal) and b (N-terminal) ions are indicated in the spectra. The ions marked with asterisks indicate the fragments produced after neutral loss of H3PO4 (98 Da). (A) The doubly charged parent ion with m/z 481.6 is labelled in the spectrum along with the ion at m/z 432.6 produced after the neutral loss of 98 Da. The shown peptide sequence revealed from the spectrum corresponds to amino acids 363–370 in PTRF. The lower-case s indicates phosphorylation of Ser-365 in PTRF. (B) The doubly charged parent ion with m/z 521.6 is labelled in the spectrum along with the ions at m/z 472.6 and 423.6 produced after the single and double neutral losses of 98 Da respectively. The peptide sequence corresponds to amino acids 363–370 in PTRF, with phosphorylation of Ser-365 and Ser-366 indicated by the lower-case s. (C) The doubly charged parent ion with m/z 819.3 is labelled in the spectrum along with the ion at m/z 770.3 produced after the neutral loss of 98 Da. The peptide sequence corresponds to amino acids 31–45 in PTRF. The lower-case s indicates phosphorylation of Ser-40 in PTRF. (D) The parent ion with m/z 859.3 is labelled in the spectrum along with the ion at m/z 810.3 produced after the neutral loss of 98 Da. The peptide sequence corresponds to amino acids 31–45 in PTRF, with phosphorylation of Ser-36 and Ser-40 indicated by the lower-case s.

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