Cloning and Expression of Palmitoyl Protein Thioesterases and Acyl Protein Thioesterases for Use in Depalmitoylation Reactions

Abstract

Palmitoylation is a dynamic process for regulating protein function and localization. It involves the attachment of palmitate to protein cysteine residues by palmitoyl transferases and its removal by palmitoyl protein thioesterase (PPTs) or acyl protein thioesterases (APTs). Techniques to study palmitoylation are difficult to perform and use harsh depalmitoylating reagents like hydroxylamine (HAM), often leading to destruction of the protein being studied. Using recombinant or over‐expressed PPT/APTs could be used as an alternative to HAM for studying protein palmitoylation/depalmitoylation dynamics, as well as investigating PPT/APT substate specificities. We have cloned human APT and PPT cDNA for the production of GST or MBP‐tagged fusion proteins to be used in acyl‐biotin exchange assays. We have also produced mCherry‐tagged versions for co‐expression with a target protein of interest to evaluate their activities. In these experiments, we will focus on the Rho GTPase TCL/RhoJ, which was recently shown to associate with the plasma membrane through palmitoylation. Cherry‐tagged APT/PPTs will be co‐transfected with YFP‐tagged TCL to determine if TCL membrane association diminishes. Overall, the goal of these experiments is to improve techniques for studying protein palmitoylation/depalmitoylation.Support or Funding InformationThis work was supported by Bemidji State University Biology Department and the College of Business, Mathematics, and Science. Support was also provided through the Neilson Foundation, Bemidji, MN and Regenerative Medicine Minnesota (RMM‐2017‐EP‐04).