Topological Analysis of Hedgehog Acyltransferase, a Multipalmitoylated Transmembrane Protein

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.Background: Hedgehog acyltransferase (HHAT) palmitoylates hedgehog proteins and is a potential target in cancer.Results: HHAT has ten transmembrane domains, two reentrant loops, and four palmitoylation sites.Conclusion: HHAT topology is determined, and protein is multipalmitoylated, which modulates protein function.Significance: Elucidating HHAT topology and posttranslational modifications is crucial to understand its acyltransferase activity and to develop new strategies to treat cancer.