Unique self-palmitoylation activity of the transport protein particle component Bet3: a mechanism required for protein stability.
Bet3 is a component of the transport protein particle complex involved in vesicular trafficking to and through the Golgi complex. X-ray structural analysis of human and mouse Bet3 revealed a hydrophobic tunnel inside the protein, which is occupied by a fatty acid linked to cysteine-68. We show here that Bet3 has strong self-palmitoylating activity. Incubation of purified Bet3 with [3H]palmitoyl-CoA (Pal-CoA) leads to a rapid and stoichiometric attachment of fatty acids to cysteine-68. Bet3 has an intrinsic affinity for Pal-CoA, and the palmitoylation reaction occurs at physiological pH values and Pal-CoA concentrations. Moreover, Bet3 is also efficiently palmitoylated at cysteine-68 inside vertebrate cells. Palmitoylation can occur late after Bet3 synthesis, but once the fatty acids are bound they are not removed, not even by disassembly of the Golgi complex. Narrowing the hydrophobic tunnel by exchange of alanine-82 with bulkier amino acids inhibits palmitoylation, both in vitro and inside cells, indicating that the fatty acid must insert into the tunnel for stable attachment. Finally, we show that palmitoylation of Bet3 plays a structural role. CD spectroscopy reveals that chemically deacylated Bet3 has a reduced melting temperature. As a consequence of its structural defect nonacylated Bet3 does not bind to TPC6, a further subunit of the transport protein particle complex, and is degraded inside cells.
Kümmel
Daniel
D
Max Delbrück Center for Molecular Medicine, Robert-Rössle Strasse 10, 13125 Berlin, Germany.
Heinemann
Udo
U
Veit
Michael
M
D000818:Animals; D001120:Arginine / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D006224:Cricetinae; D003545:Cysteine / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008958:Models, Molecular; D019308:Palmitic Acid / Q000737:chemistry* / Q000378:metabolism*; D010171:Palmitoyl Coenzyme A / Q000378:metabolism; D011485:Protein Binding; D011489:Protein Denaturation; D017434:Protein Structure, Tertiary; D013696:Temperature; D033921:Vesicular Transport Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
C503571:TRAPPC3 protein, human; D033921:Vesicular Transport Proteins; D010171:Palmitoyl Coenzyme A; D019308:Palmitic Acid; D001120:Arginine; D003545:Cysteine