Crystal structure of bet3 reveals a novel mechanism for Golgi localization of tethering factor TRAPP

Abstract

Transport protein particle (TRAPP) is a large multiprotein complex involved in endoplasmic reticulum-to-Golgi and intra-Golgi traffic. TRAPP specifically and persistently resides on Golgi membranes. Neither the mechanism of the subcellular localization nor the function of any of the individual TRAPP components is known. Here, the crystal structure of mouse Bet3p (bet3), a conserved TRAPP component, reveals a dimeric structure with hydrophobic channels. The channel entrances are located on a putative membrane-interacting surface that is distinctively flat, wide and decorated with positively charged residues. Charge-inversion mutations on the flat surface of the highly conserved yeast Bet3p led to conditional lethality, incorrect localization and membrane trafficking defects. A channel-blocking mutation led to similar defects. These data delineate a molecular mechanism of Golgi-specific targeting and anchoring of Bet3p involving the charged surface and insertion of a Golgi-specific hydrophobic moiety into the channels. This essential subunit could then direct other TRAPP components to the Golgi.

Unique self-palmitoylation activity of the transport protein particle component Bet3: A mechanism required for protein stability

“…Bet3 is the only known eukaryotic protein containing covalently bound fatty acids when purified from E. coli, which lack enzymes for palmitoylation (8). Thus, even in cells acylation of Bet3 does not require an exogenous enzyme.…”

“…3A), indicating that the fatty acid must insert into the tunnel to become stably attached. Multiple sequence alignment of Bet3 sequences showed adjacent to the palmitoylated cysteine an arginine, which is completely conserved from yeast to man (8,9). Basic amino acids in the vicinity of palmitoylated cysteines often affect the acylation reaction, probably by decreasing the pK a of the cysteine's -sulfhydryl group (19,20).…”

“…Instead, an acyl chain linked to a Golgi-resident membrane protein was proposed to insert into the hydrophobic tunnel, thereby promoting membrane binding of Bet3. The same deleterious effects on yeast survival and vesicular trafficking were noticed if positively charged amino acids on a flat surface of Bet3 were substituted with negatively charged residues, suggesting that they also contribute to membrane binding of Bet3 (8). However, overexpression of Trs33 (yeast homolog of TPC6) was able to restore growth of these mutants (11).…”

“…The Bet3-TPC6 subcomplex is thought to represent the core of TRAPP and to be involved in anchoring the complex to the Golgi membrane. Surprisingly, the crystals of Bet3 revealed a long-chain fatty acid covalently bound via a thioester linkage to the conserved residue cysteine-68, even if the recombinant protein used for crystallization was purified from Escherichia coli, which lack enzymes for acylation of proteins (8,9). The carbon chain is not exposed at the surface of the molecule but is completely buried inside a hydrophobic tunnel.…”

“…X-ray crystallography with Bet3 from mice and humans revealed a novel dimeric protein fold, which is also present in a second TRAPP subunit, TPC6 (8)(9)(10)(11)(12). Biochemical and crystallographic studies showed that Bet3 and TPC6 form heterodimers that are likely to be functional modules of TRAPP.…”