title: Membrane topology of hedgehog acyltransferase.
abstract: Hedgehog acyltransferase (Hhat) is a multipass transmembrane enzyme that mediates the covalent attachment of the 16-carbon fatty acid palmitate to the N-terminal cysteine of Sonic Hedgehog (Shh). Palmitoylation of Shh by Hhat is critical for short and long range signaling. Knowledge of the topological organization of Hhat transmembrane helices would enhance our understanding of Hhat-mediated Shh palmitoylation. Bioinformatics analysis of transmembrane domains within human Hhat using 10 different algorithms resulted in highly consistent predictions in the C-terminal, but not in the N-terminal, region of Hhat. To empirically determine the topology of Hhat, we designed and exploited Hhat constructs containing either terminal or 12 different internal epitope tags. We used selective permeabilization coupled with immunofluorescence as well as a protease protection assay to demonstrate that Hhat contains 10 transmembrane domains and 2 re-entrant loops. The invariant His and highly conserved Asp residues within the membrane-bound O-acyltransferase (MBOAT) homology domain are segregated on opposite sides of the endoplasmic reticulum membrane. The localization of His-379 on the lumenal membrane surface is consistent with a role for this invariant residue in catalysis. Analysis of the activity and stability of the Hhat constructs revealed that the C-terminal MBOAT domain is especially sensitive to manipulation. Moreover, there was remarkable similarity in the overall topological organization of Hhat and ghrelin O-acyltransferase, another MBOAT family member. Knowledge of the topological organization of Hhat could serve as an important tool for further design of selective Hhat inhibitors.
authors.affiliation: From the Cell Biology Program, the Gerstner Sloan Kettering Graduate School of Biomedical Sciences, Memorial Sloan Kettering Cancer Center, New York, New York 10065.
authors.forename: Armine
authors.initials: A
authors.lastname: Matevossian
authors.affiliation: From the Cell Biology Program, the Gerstner Sloan Kettering Graduate School of Biomedical Sciences, Memorial Sloan Kettering Cancer Center, New York, New York 10065 reshm@mskcc.org.
authors.forename: Marilyn D
authors.initials: MD
authors.lastname: Resh
mesh_terms: D000217:Acyltransferases / Q000378:metabolism*; D000818:Animals; D019556:COS Cells; D002384:Catalysis; D002522:Chlorocebus aethiops; D019295:Computational Biology; D004721:Endoplasmic Reticulum / Q000378:metabolism; D000939:Epitopes / Q000378:metabolism; D019084:Fluorescent Antibody Technique, Indirect; D053823:Hedgehog Proteins / Q000378:metabolism*; D006801:Humans; D054878:Lipoylation; D008856:Microscopy, Fluorescence; D010168:Palmitates / Q000378:metabolism*; D010957:Plasmids / Q000378:metabolism; D017434:Protein Structure, Tertiary; D015398:Signal Transduction
chemical_list: D000939:Epitopes; D053823:Hedgehog Proteins; D010168:Palmitates; D000217:Acyltransferases; C542776:MBOAT4 protein, human; C105599:HHAT protein, human
keywords: Membrane Bilayer; Membrane Protein; Protein Palmitoylation; Sonic Hedgehog (SHH); Transmembrane Domain