Protein fatty acylation is the attachment of a long-chain fatty acyl group, generally 14–16 carbons in length to the side chain of an amino acid. Cysteine palmitoylation or S-palmitoylation is best understood and widespread post-translational modification in this class, followed closely by N-terminal glycine myristoylation and lastly by O-serine fatty acylation. In recent years, a previously underappreciated member of this class, lysine N ε - fatty acylation, has gained traction due to work by Jiang et al with their discovery that SIRT6 can defatty-acylate TNFα and regulate its secretion, providing the first example for the function of protein lysine fatty acylation in mammalian cells 1,2 . Additional substrates such as K-Ras4a, and RRAS2 further highlight the importance of this modification. However, while advances have been made in the identification and study of the erasers of this modification, little progress has been made in the identification of the writers of this modification. The goal of my thesis project is to identify the writers of lysine fatty acylation. We report the identification of the first mammalian N ε -lysine fatty acyl transferase. We identified the writer through screening various acyl transferases characterized as S-palmitoyl transferases, O-serine fatty acyl transferases, and lipid fatty acyl transferases, against substrates identified to have lysine fatty acylation. Multiple family members of the DHHC class of S- palmitoyl transferases were identified as lysine fatty acyl transferases. Utilizing alkynyl-fatty acid labeling with in-gel fluorescence, we observed members DHHC2/3/7/15 of the DHHC family of S-palmitoyl transferase labeling the lysine residues of multiple GTPases. This acylation event is confirmed as lysine fatty acylation through the use of the SIRT2 in-vitro defatty-acylation assay and mutagenesis of the modification sites and P-32 NAD + assay. Knockdown of the zDHHC7 gene was able to perturb the lysine fatty acylation of KRAS4A. Screening additional GTPases, not known to have lysine fatty acylation, against DHHC7 identified additional substrates such as CDC42. We identified DHHC7 mediated lysine fatty acylation of CDC42 inactivates the GTPase and induces morphological change in a similar manner as a CDC42 inactive mutant. Expression of a lysine mutant of CDC42 which cannot be acylated by DHHC7 is able to rescue the cell and prevent DHHC7 induced cell rounding. The discovery of the novel lysine acyltransferase activity of the DHHC enzymes highlights the importance of lysine fatty acylation and provides insights to further understanding the function of DHHC enzymes and protein lysine fatty acylation.