Neuronal palmitoyl acyl transferases exhibit distinct substrate specificity.
Palmitoylation, a post-translational modification of cysteine residues with the lipid palmitate, has recently emerged as an important mechanism for regulating protein trafficking and function. With the identification of 23 DHHC mammalian palmitoyl acyl transferases (PATs), a key question was the nature of substrate-enzyme specificity for these PATs. Using the acyl-biotin exchange palmitoylation assay, we compared the substrate specificity of four neuronal PATs, namely DHHC-3, DHHC-8, HIP14L (DHHC-13), and HIP14 (DHHC-17). Exogenous expression of enzymes and substrates in COS cells reveals that HIP14L and HIP14 modulate huntingtin palmitoylation, DHHC-8 modulates paralemmin-1 palmitoylation, and DHHC-3 shows the least substrate specificity. These in vitro data were validated by lentiviral siRNA-mediated knockdown of endogenous HIP14 and DHHC-3 in cultured rat cortical neurons. PATs require the presence of palmitoylated cysteines in order to interact with their substrates. To understand the elements that influence enzyme/substrate specificity further, we fused the HIP14 ankryin repeat domain to the N terminus of DHHC-3, which is not a PAT for huntingtin. This modification enabled DHHC-3 to behave similarly to HIP14 by modulating palmitoylation and trafficking of huntingtin. Taken together, this study indicates that individual PATs have specific substrate preference, determined by regulatory domains outside the DHHC domain of the enzymes.
Huang
Kun
K
Centre for Molecular Medicine and Therapeutics, University of British Columbia, Vancouver, BC V5Z 4H4, Canada.
Sanders
Shaun
S
Singaraja
Roshni
R
Orban
Paul
P
Cijsouw
Tony
T
Arstikaitis
Pamela
P
Yanai
Anat
A
Hayden
Michael R
MR
El-Husseini
Alaa
A
D000217:Acyltransferases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D006056:Golgi Apparatus / Q000201:enzymology; D000071058:Huntingtin Protein; D066298:In Vitro Techniques; D054878:Lipoylation / Q000502:physiology*; D008565:Membrane Proteins / Q000378:metabolism; D008958:Models, Molecular; D009419:Nerve Tissue Proteins / Q000378:metabolism; D009474:Neurons / Q000201:enzymology*; D009687:Nuclear Proteins / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
C499524:Htt protein, rat; D000071058:Huntingtin Protein; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D009687:Nuclear Proteins; C523858:Palm protein, rat; D010750:Phosphoproteins; D034741:RNA, Small Interfering; D011994:Recombinant Proteins; D000217:Acyltransferases