期刊
CELLULAR AND MOLECULAR LIFE SCIENCES
卷 67, 期 15, 页码 2653-2664出版社
SPRINGER BASEL AG
DOI: 10.1007/s00018-010-0358-y
关键词
Golgi; Vesicular transport; Tethering; TRAPP; Bet3; Palmitoylation; Acyl-CoA
资金
- DFG [SFB 740, TP C1]
- Fonds der Chemischen Industrie
- Medical Research Council [G0500367] Funding Source: researchfish
- MRC [G0500367] Funding Source: UKRI
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.
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