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 alpha 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.