Emerging evidence suggests that ubiquitination serves as a protein trafficking signal in addition to its well characterized role in promoting protein degradation. The yeast G protein α subunit Gpa1 represents a rare example of a protein that undergoes both mono- and poly-ubiquitination. Whereas mono-ubiquitinated Gpa1 is targeted to the vacuole, poly-ubiquitinated Gpa1 is directed instead to the proteasome. Here we investigate the structural requirements for mono- and poly-ubiquitination of Gpa1. We find that variants of Gpa1 engineered to be unstable are more likely to be poly-ubiquitinated and less likely to be mono-ubiquitinated. In addition, mutants that cannot be myristoylated are no longer mono-ubiquitinated but are still polyubiquitinated. Finally, we show that the ubiquitin ligase Rsp5 is necessary for Gpa1 mono-ubiquitination in vivo and that the purified enzyme is sufficient to catalyze Gpa1 mono-ubiquitination in vitro. Taken together, these data indicate that mono- and poly-ubiquitination have distinct enzyme and substrate recognition requirements; whereas poly-ubiquitination targets misfolded protein for degradation, a distinct ubiquitination apparatus targets the fully mature, fully myristoylated G protein for mono-ubiquitination and delivery to the vacuole.