Hhat Is a Palmitoylacyltransferase with Specificity for N-Palmitoylation of Sonic Hedgehog

John A Buglino,Marilyn D Resh

doi:10.1074/jbc.m803901200

Abstract

Palmitoylation of Sonic Hedgehog (Shh) is critical for effective long- and short-range signaling. Genetic screens uncovered a potential palmitoylacyltransferase (PAT) for Shh, Hhat, but the molecular mechanism of Shh palmitoylation remains unclear. Here, we have developed and exploited an in vitro Shh palmitoylation assay to purify Hhat to homogeneity. We provide direct biochemical evidence that Hhat is a PAT with specificity for attaching palmitate via amide linkage to the N-terminal cysteine of Shh. Other palmitoylated proteins (e.g. PSD95 and Wnt) are not substrates for Hhat, and Porcupine, a putative Wnt PAT, does not palmitoylate Shh. Neither autocleavage nor cholesterol modification is required for Shh palmitoylation. Both the Shh precursor and mature protein are N-palmitoylated by Hhat, and the reaction occurs during passage through the secretory pathway. This study establishes Hhat as a bona fide Shh PAT and serves as a model for understanding how secreted morphogens are modified by distinct PATs.

Highlights

Hedgehog (Hh) and Sonic Hedgehog (Shh) are members of a family of secreted signaling proteins that mediate growth and patterning during embryonic development [1, 2]
Reconstitution of Shh Palmitoylation in Vivo—Given the inherent difficulty involved in purifying polytopic membrane proteins in an active conformation, we first sought to reconstitute the Shh palmitoylation reaction in tissue culture cells
COS-1 cells were cotransfected with plasmids encoding fulllength Shh and either empty pcDNA3.1 vector or HA-tagged Hhat in pcDNA3.1

Summary

EXPERIMENTAL PROCEDURES

Reagents and Antibodies—Fatty acyl-CoAs, CoA, CoA synthetase, octyl glucoside, anti-FLAG and anti-hemagglutinin (HA) antibodies, FLAG M2-agarose, and 3XFLAG peptide were purchased from Sigma. The supernatant was discarded, and the pellets were resuspended in 10 ml of solubilization/wash buffer (20 mM HEPES (pH 7.3), 350 mM NaCl, 1% octyl glucoside, and 1% glycerol) and incubated on ice for 1 h, followed by centrifugation at 100,000 ϫ g. In Vitro Palmitoylation Assay—The in vitro assay was performed by incubating 10 ␮l of Hhat-HA-FLAG-His in 20 mM HEPES (pH 7.3), 100 mM NaCl, 1% octyl glucoside, and 1% glycerol with 10 ␮l of recombinant Shh (0.2– 0.4 mg/ml in 20 mM MES (pH 6.5), 1 mM EDTA, and 1 mM DTT), followed by the addition of 30 ␮l of reaction buffer (167 mM MES (pH 6.5), 1.7 mM DTT, 0.083% Triton X-100, and 167 ␮M [125I]iodopalmitoyl-CoA). Samples were incubated in either 1 M Tris (pH 8.0) or hydroxylamine (pH 8.0) for 1 h at room temperature, followed by two washes with RIPA buffer

RESULTS

Palmitoylation of Shh in Hhattransfected Cells Occurs via Amide

Specific activity

Kinetic measurements

DISCUSSION