Topological analysis of Hedgehog acyltransferase, a multi-palmitoylated transmembrane protein [Cell Biology]

December 12th, 2014 by Konitsiotis, A. D., Jovanović, B., Ciepla, P., Spitaler, M., Lanyon–Hogg, T., Tate, E. W., Magee, A. I.

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 localisation. Hedgehog acyltransferase (HHAT) is the enzyme in the endoplasmatic reticulum (ER) that palmitoylates Hedgehog proteins, is a member of a small subfamily of MBOAT proteins that acylate secreted proteins, and is an important drug target in cancer. However little is known about HHATs structure and mode of function. We show that HHAT is comprised of 10 transmembrane domains and 2 reentrant loops with the critical His and Asp residues on opposite sides of the ER membrane. We further show that HHAT is palmitoylated on multiple cytosolic cysteines, which maintain protein structure within the membrane. Finally, we provide evidence that mutation of the conserved His residue in the hypothesised catalytic domain results in a complete loss of HHAT palmitoylation, providing novel insight into how the protein may function in vivo.