Characterization of S-palmitoylation dependent protein localization by hyperplexed spatial proteomics
Summary
S-Palmitoylation is a post translational modification (PTM) associated with numerous cancers and
neurological diseases. It is a reversible type of lipidation defined by the addition of a palmitic acid to a
cysteine residue. Regulation of this PTM depends on the activity of palmitoyl S-acyl transferases and
palmitoyl thioesterases which add and remove the palmitoyl moiety, respectively. S-palmitoylation can
have various effects on proteins, of which a change in intracellular localization is most substantiated.
While methods for large scale identification of palmitoylated proteins have become well established, the
characterization of S-palmitoylation dependent translocations has so far only been studied on a single
protein basis. This study aimed to identify S-palmitoylation dependent translocating proteins. The broad
spectrum depalmitoylase inhibitor Palmostatin B (PalmB) was employed in combination with differential
centrifugation based spatial proteomics to determine translocating proteins. In addition, SILAC-TMT
hyperplexing was used to allow for the combined quantitation of treatment groups and spatial
proteomics fractions. Using this method we identified 48 potentially translocating proteins. A high
overrepresentation of S-palmitoylated proteins was observed among the translocators indicating that
the translocation of these enriched proteins may have occurred in response to a change in
palmitoylation. Additionally, a STRING database search found many interacting proteins within this
group suggesting that some translocations may occur as a result of the translocation of a binding
partner. We demonstrate that spatial proteomics can be used as a hypothesis-generating method to
identify potential S-palmitoylation dependent translocating proteins. These potential translocators can
be used as a starting point for further study into S-palmitoylation dependent protein localization.