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Hedgehog proteins are secreted morphogens that play critical roles in development and disease. During maturation of the proteins through the secretory pathway, they are modified by the addition of N-terminal palmitic acid and C-terminal cholesterol moieties, both of which are critical for their correct function and localization. Hedgehog acyltransferase (HHAT) is the enzyme in the endoplasmic reticulum that palmitoylates Hedgehog proteins, is a member of a small subfamily of membrane-bound O-acyltransferase proteins that acylate secreted proteins, and is an important drug target in cancer. However, little is known about HHAT structure and mode of function. We show that HHAT is comprised of ten transmembrane domains and two reentrant loops with the critical His and Asp residues on opposite sides of the endoplasmic reticulum membrane. We further show that HHAT is palmitoylated on multiple cytosolic cysteines that maintain protein structure within the membrane. Finally, we provide evidence that mutation of the conserved His residue in the hypothesized catalytic domain results in a complete loss of HHAT palmitoylation, providing novel insights into how the protein may function in vivo.

Original publication

DOI

10.1074/jbc.M114.614578

Type

Journal article

Journal

J Biol Chem

Publication Date

2015

Volume

290

Pages

3293 - 3307

Keywords

Acyltransferases/*chemistry/genetics/metabolism Amino Acid Motifs *Catalytic Domain HEK293 Cells HeLa Cells Humans Lipoylation Mutation *Protein Processing, Post-Translational Acyltransferase Cancer Click Chemistry Hhat Hedgehog Signaling Pathway Mboat Protein Palmitoylation Topology Transmembrane Domain