Protein prenylation is the irreversible attachment of an isoprenoid lipid moiety near the C-terminus of a protein, which can facilitate membrane localization and protein-protein interactions. Prenylated proteins are part of many signaling pathways and are involved in the development of many diseases, including various cancers and progeria. Protein farnesyltransferase (FTase) and geranylgeranyltransferase-I (GGTase-I) catalyze prenylation by attaching a 15- (farnesyl) or 20-carbon (geranylgeranyl) isoprenoid, respectively, to a cysteine four residues from the C-terminus of a protein (-CaaX). Prenylated proteins are often further processed by proteolysis of the -aaX sequence and carboxymethylation. FTase and GGTase-I are cytosolic enzymes whereas downstream processing enzymes are embedded in the ER and nuclear membranes; it is unclear how prenylated proteins traffic to these downstream enzymes. Proteins containing CaaX sequences were thought to be constitutively prenylated without cellular regulation, however recent data suggest entry and trafficking through the prenylation pathway is regulated. Two SmgGDS splice variants, with different binding preferences and functions, have been proposed to regulate prenylation. SmgGDS-607 is proposed to specifically interact with nonprenylated small GTPases and regulate their entry into the prenylation pathway whereas SmgGDS-558 interacts with prenylated small GTPases and facilitates trafficking. These hypotheses will be tested by defining SmgGDS isoform binding preferences using in vitro pulldown assays and mutagenesis to analyze the importance of the following GTPase structural elements for determining binding affinity: prenyl-group identity, CaaX sequence, and a polybasic sequence. The ability of SmgGDS isoforms to regulate prenylation catalyzed by FTase and GGTase-I will be tested using either peptides or full-length proteins, and the mechanisms of regulation will be determined. To better understand how CaaX proteins traffic from the cytosol to membranes, an innovative approach of microinjection and live cell imaging will be used. Intracellular Single-molecule High-Resolution Localization and Counting (iSHiRLoC) enables the synchronized initiation of proteins into cellular processing pathways and tracking their cellular location, measurement of trafficking rates, and the visualization of rare events. iSHiRLoC will be used to characterize the movement of various CaaX proteins through the prenylation pathway and the influence of prenylation status and isoprenoid identity on trafficking rates will be determined. Small interfering RNA treatment prior to iSHiRLoC will enable determination of the role that specific proteins, such as SmgGDS isoforms and another chaperones PDE6?, have in regulating cellular prenylation and trafficking dynamics. Small GTPases associated with diseases will be selected for both in vitro and in vivo studies. Therefore, knowledge gained will provide insight into developing therapeutic approaches for treating diseases as well as expanding our understanding of the regulation and trafficking of lipidated proteins.