Proteomic analysis of membrane-associated proteins from rat liver autophagosomes.

Proteins associated with membranes from purified rat liver autophagosomes were separated by two-dimensional (2D) gel electrophoresis (zoom gels, pl 4-7 and 6-9), silver-stained and identified by MALDI-TOF mass spectrometry. Among >1,500 detectable protein spots, 58 (derived from 39 different known proteins) were at least twofold (and significantly) enriched in autophagosomal membranes relative to cytoplasmic membranes. All of these membrane-associated proteins were also present in the cytosol, many of them being truncated enzyme variants that would be expected to serve a binding rather than an enzymatic function. Eleven proteins were highly enriched (consistent with the theoretical maximum of 25x), corresponding to an exclusive membrane localization in the delimiting membrane of the autophagosome. Three of these were methyltransferases: betaine:homocysteine methyltransferase (five variants); catechol O-methyltransferase (one phosphorylated and one unphosphorylated variant) and methionine adenosyltransferase, perhaps indicating that methylation/demethylation of membrane components could play a role in autophagy. A fourth highly enriched autophagosomal protein, phosphatidylethanolamine binding protein, is particularly interesting considering that the autophagic marker protein, LC3/ Atg8, is linked to autophagosomal membranes through its covalent conjugation with phosphatidylethanolamine (as the form LC3-II). LC3-II was not detectable on silver-stained 2D-gels, but could be shown by immunoblotting to be highly enriched in autophagosomal membranes. Other highly enriched proteins were heat shock cognate protein Hsc70 (one short and one long variant), peroxiredoxin 2, peroxiredoxin 6 (two variants), fructose 1,6-bisphosphatase (one phosphorylated and one unphosphorylated variant), adenosine kinase, inorganic pyrophosphatase and selenium-binding protein 2. Hsc70, a chaperonin that plays an important role in the recognition and proteasomal degradation of aggregated proteins as well as in the lysosomal membrane uptake and degradation of certain cytosolic proteins (chaperone-mediated autophagy), could conceivably also serve a recognition function in the autophagic scavenging of denatured or aggregated proteins (aggrephagy). The moderately enriched (2-14x) autophagosomal membrane-associated proteins included a remarkably high proportion of drug-metabolizing enzymes, such as several glutathione S-transferases, sulfotransferases and aromatic hydrocarbon/steroid oxidoreductases. If the autophagic function of these proteins is to recognize protein-drug adducts, they may, along with the peroxiredoxins, chaperonins and methyl metabolic enzymes, make the phagophores (the sequestering precursors of the autophagosomal delimiting membrane) well equipped for the detection and scavenging of proteins denatured by oxidation, hypermethylation, drug adduction or other mechanisms.

Comigration of two autophagosome-associated dehydrogenases on two-dimensional polyacrylamide gels.

Immunoblotting of two-dimensional polyacrylamide gels (pI 3-10) revealed six cytosolic molecular forms of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in rat hepatocytes. Two of the four full-length (approximately 37 kDa) forms exhibited some binding to sedimentable cellular elements (but not to mitochondria), whereas one full-length and two short (approximately 35 kDa) forms selectively bound to the membranes of autophagosomes and lysosomes. Tryptic fingerprinting by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) confirmed the identity of the major full-length forms as GAPDH, but attempts to identify the major short form consistently suggested that this spot represented a different enzyme, 3-alpha-hydroxysteroid dehydrogenase (3alphaHSD). Silver staining indicated that this 3alphaHSD form selectively bound to autophagosomal and lysosomal membranes. Immunoblotting of more focused 2D gels (pI 6-9) with an antibody raised against 3alphaHSD demonstrated immunostaining of four 3alphaHSD forms with masses of about 35 kDa. Autophagosomal membrane preparations were highly and selectively enriched with respect to all of these 3alphaHSD forms. One of them comigrated with the major short form of GAPDH, accounting for the paradoxical mass spectrometric identification of 3alphaHSD from this spot. Proteomic analysis by a combination of immunological and mass spectrometric identification methods was thus capable of resolving two comigrating dehydrogenases selectively associated with autophagic organelles.

Okadaic acid-induced, naringin-sensitive phosphorylation of glycine N-methyltransferase in isolated rat hepatocytes.

Glycine N-methyltransferase (GNMT) is an abundant cytosolic enzyme that catalyses the methylation of glycine into sarcosine, coupled with conversion of the methyl donor, S -adenosylmethionine (AdoMet), into S -adenosylhomocysteine (AdoHcy). GNMT is believed to play a role in monitoring the AdoMet/AdoHcy ratio, and hence the cellular methylation capacity, but regulation of the enzyme itself is not well understood. In the present study, treatment of isolated rat hepatocytes with the protein phosphatase inhibitor okadaic acid, was found to induce an overphosphorylation of GNMT, as shown by proteomic analysis. The analysis comprised two-dimensional gel electrophoretic separation of (32)P-labelled phosphoproteins and identification of individual protein spots by matrix-assisted laser-desorption ionization-time-of-flight mass spectrometry. The identity of GNMT was verified by N-terminal Edman sequencing of tryptic peptides. Chromatographic separation of proteolytic peptides and (32)P-labelled amino acids suggested that GNMT was phosphorylated within a limited region, and only at serine residues. GNMT phosphorylation could be suppressed by naringin, an okadaic acid-antagonistic flavonoid. To assess the possible functional role of GNMT phosphorylation, the effect of okadaic acid on hepatocytic AdoMet and AdoHcy levels was examined, using HPLC separation for metabolite analysis. Surprisingly, okadaic acid was found to have no effect on the basal levels of AdoMet or AdoHcy. An accelerated AdoMet-AdoHcy flux, induced by the addition of methionine (1 mM), was likewise unaffected by okadaic acid. 5-Aminoimidazole-4-carboxamide riboside, an activator of the hepatocytic AMP-activated protein kinase, similarly induced GNMT phosphorylation without affecting AdoMet and AdoHcy levels. Activation of cAMP-dependent protein kinase by dibutyryl-cAMP, reported to cause GNMT phosphorylation under cell-free conditions, also had little effect on hepatocytic AdoMet and AdoHcy levels. Phosphorylation of GNMT would thus seem to play no role in regulation of the intracellular AdoMet/AdoHcy ratio, but could be involved in other GNMT functions, such as the binding of folates or aromatic hydrocarbons.