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    Characterization of the self-palmitoylation activity of the transport protein particle component Bet3 (2010)

    Art
    Zeitschriftenartikel / wissenschaftlicher Beitrag
    Autoren
    Kümmel, Daniel
    Walter, Julia
    Heck, Martin
    Heinemann, Udo
    Veit, Michael
    Quelle
    Cellular and molecular life sciences : CMLS; 67(15) — S. 2653–2664
    ISSN: 1420-682x
    Sprache
    Englisch
    Verweise
    DOI: 10.1007/s00018-010-0358-y
    Pubmed: 20372964
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    Abstract / Zusammenfassung

    Bet3, a transport protein particle component involved in vesicular trafficking, contains a hydrophobic tunnel occupied by a fatty acid linked to cysteine 68. We reported that Bet3 has a unique self-palmitoylating activity. Here we show that mutation of arginine 67 reduced self-palmitoylation of Bet3, but the effect was compensated by increasing the pH. Thus, arginine helps to deprotonate cysteine such that it could function as a nucleophile in the acylation reaction which is supported by the structural analysis of non-acylated Bet3. Using fluorescence spectroscopy we show that long-chain acyl-CoAs bind with micromolar affinity to Bet3, whereas shorter-chain acyl-CoAs do not interact. Mutants with a deleted acylation site or a blocked tunnel bind to Pal-CoA, only the latter with slightly reduced affinity. Bet3 contains three binding sites for Pal-CoA, but their number was reduced to two in the mutant with an obstructed tunnel, indicating that Bet3 contains binding sites on its surface.