Cardiolipin defines the interactome of the major ADP/ATP carrier protein of the mitochondrial inner membrane

Defined mutations in the mitochondrial ADP/ATP carrier (AAC) are associated with certain types of progressive external ophthalmoplegia. AAC is required for oxidative phosphorylation (OXPHOS), and dysregulation of AAC has been implicated in apoptosis. Little is known about the AAC interactome, aside from a known requirement for the phospholipid cardiolipin (CL) and that it is thought to function as a homodimer. Using a newly developed dual affinity tag, we demonstrate that yeast AAC2 physically participates in several protein complexes of distinct size and composition. The respiratory supercomplex and several smaller AAC2-containing complexes, including other members of the mitochondrial carrier family, are identified here. In the absence of CL, most of the defined interactions are destabilized or undetectable. The absence of CL and/or AAC2 results in distinct yet additive alterations in respiratory supercomplex structure and respiratory function. Thus, a single lipid can significantly alter the functional interactome of an individual protein

Studies of biological active peptides (structure determination, synthesis and activity) / Pinmanee Boontheung.

Includes author's previously published papers.Includes appendix in back pocket."January, 2003"Includes bibliographical references.xvi, 199 leaves, [23] : ill. (some col.), maps, plates (col.), photographs (col.) ; 30 cm.Thesis (Ph.D.)--University of Adelaide, School of Chemistry and Physics, Discipline of Chemistry, 200

Cardiolipin defines the interactome of the major ADP/ATP carrier protein of the mitochondrial inner membrane.

Defined mutations in the mitochondrial ADP/ATP carrier (AAC) are associated with certain types of progressive external ophthalmoplegia. AAC is required for oxidative phosphorylation (OXPHOS), and dysregulation of AAC has been implicated in apoptosis. Little is known about the AAC interactome, aside from a known requirement for the phospholipid cardiolipin (CL) and that it is thought to function as a homodimer. Using a newly developed dual affinity tag, we demonstrate that yeast AAC2 physically participates in several protein complexes of distinct size and composition. The respiratory supercomplex and several smaller AAC2-containing complexes, including other members of the mitochondrial carrier family, are identified here. In the absence of CL, most of the defined interactions are destabilized or undetectable. The absence of CL and/or AAC2 results in distinct yet additive alterations in respiratory supercomplex structure and respiratory function. Thus, a single lipid can significantly alter the functional interactome of an individual protein

(A–C) After CNAP, the final concentrated eluates were resolved in the first dimension by 6–16% BN-PAGE and in the second dimension by 12–18% SDS-PAGE

SYPRO Ruby detected bands were extracted, digested with trypsin, and proteins identified by LC–MS/MS. Respiratory complex III and IV subunits are labeled in red and blue, respectively, and members of the mitochondrial carrier family in purple. The pink circle highlights contaminating catalase (verified by LC–MS/MS) from the BN-PAGE molecular weight markers. (D–F) After CNAP, the final concentrated eluates were resolved by 2D BN/SDS-PAGE and immunoblotted for the indicated mitochondrial proteins. = 3. The sources of the purified extracts were (A and D) Δ[AAC2], (B and E) Δ[CNAPAAC2], and (C and F) ΔΔ[CNAPAAC2].<p><b>Copyright information:</b></p><p>Taken from "Cardiolipin defines the interactome of the major ADP/ATP carrier protein of the mitochondrial inner membrane"</p><p></p><p>The Journal of Cell Biology 2008;182(5):937-950.</p><p>Published online 8 Sep 2008</p><p>PMCID:PMC2528576.</p><p></p

Mining proteomic data to expose protein modifications in Methanosarcina mazei strain Gö1.

Proteomic tools identify constituents of complex mixtures, often delivering long lists of identified proteins. The high-throughput methods excel at matching tandem mass spectrometry data to spectra predicted from sequence databases. Unassigned mass spectra are ignored, but could, in principle, provide valuable information on unanticipated modifications and improve protein annotations while consuming limited quantities of material. Strategies to "mine" information from these discards are presented, along with discussion of features that, when present, provide strong support for modifications. In this study we mined LC-MS/MS datasets of proteolytically-digested concanavalin A pull down fractions from Methanosarcina mazei Gö1 cell lysates. Analyses identified 154 proteins. Many of the observed proteins displayed post-translationally modified forms, including O-formylated and methyl-esterified segments that appear biologically relevant (i.e., not artifacts of sample handling). Interesting cleavages and modifications (e.g., S-cyanylation and trimethylation) were observed near catalytic sites of methanogenesis enzymes. Of 31 Methanosarcina protein N-termini recovered by concanavalin A binding or from a previous study, only M. mazei S-layer protein MM1976 and its M. acetivorans C2A orthologue, MA0829, underwent signal peptide excision. Experimental results contrast with predictions from algorithms SignalP 3.0 and Exprot, which were found to over-predict the presence of signal peptides. Proteins MM0002, MM0716, MM1364, and MM1976 were found to be glycosylated, and employing chromatography tailored specifically for glycopeptides will likely reveal more. This study supplements limited, existing experimental datasets of mature archaeal N-termini, including presence or absence of signal peptides, translation initiation sites, and other processing. Methanosarcina surface and membrane proteins are richly modified