Characterization of the self-palmitoylation activity of the transport protein particle component Bet3.
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.
Kümmel
Daniel
D
Max Delbrück Center for Molecular Medicine, Robert-Rössle-Strasse 10, Berlin, Germany.
Walter
Julia
J
Heck
Martin
M
Heinemann
Udo
U
Veit
Michael
M
D000214:Acyl Coenzyme A / Q000235:genetics / Q000378:metabolism; D000215:Acylation; D001120:Arginine / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites / Q000235:genetics; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D003545:Cysteine / Q000235:genetics / Q000378:metabolism; D006056:Golgi Apparatus / Q000235:genetics / Q000378:metabolism; D054878:Lipoylation
D000214:Acyl Coenzyme A; D002352:Carrier Proteins; D001120:Arginine; D003545:Cysteine