The MHC class I antigen presentation pathway is a frequent target of virus-encoded immune evasion molecules. Proteins of the viral K3 family are E3 ubiquitin ligases that downregulate immune modulatory molecules. Further, it has been established that the K3 family has cellular homologs (Membrane-Associated RING-CH proteins), some of which act to regulate immune molecules via ubiquitination. The mK3 molecule of γ-herpesvirus 68 has been shown to utilize ER-associated degradation pathways to degrade MHC class I, thereby inhibiting antigen presentation. Here, we have explored the basis for specific targeting of class I for ubiquitination by mK3. We provide direct evidence that adapter proteins (TAP/tapasin, in this case) in the ER membrane position the RING-CH domain of mK3 to permit ubiquitination of the cytosolic tail of the class I heavy-chain. This was accomplished by generating a novel substrate for mK3 which could be ubiquitinated only when placed within the proper orientation within the TAP complex. These findings demonstrate an essential role of adapter proteins in ubiquitin ligase substrate selection, with implications for substrate selection by other K3-related E3 ligases within the immune system.