Nonsense-mediated mRNA decay (NMD) is a surveillance mechanism that accelerates 
the degradation of mRNAs containing premature translation termination codons. 
This quality control pathway depends on the NMD-specific factors, Upf1p, 
Upf2p/Nmd2p, and Upf3p, as well as the two release factors, eRF1 and eRF3 
(respectively designated Sup45p and Sup35p in yeast). NMD activation is also 
enabled by the absence of the poly(A)-binding protein, Pab1p, downstream of a 
termination event. Since Sup35p interacts with both Upf1p and Pab1p we 
considered the possibility that differential binding of the latter factors to 
Sup35p may be a critical determinant of NMD sensitivity for an mRNA. Here we 
describe three approaches to assess this hypothesis. First, we tethered 
fragments or mutant forms of Sup35p downstream of a premature termination codon 
in a mini-pgk1 nonsense-containing mRNA and showed that the inhibition of NMD by 
tethered Sup35p does not depend on the domain necessary for the recruitment of 
Pab1p. Second, we examined the Sup35p interaction properties of Upf1p and Pab1p 
in vitro and showed that these two proteins bind differentially to Sup35p. 
Finally, we examined competitive binding between the three proteins and observed 
that Upf1p inhibits Pab1p binding to Sup35p whereas the interaction between 
Upf1p and Sup35p is relatively unaffected by Pab1p. These data indicate that the 
binding of Upf1p and Pab1p to Sup35p may be more complex than anticipated and 
that NMD activation could involve more than just simple competition between 
these factors. We conclude that activation of NMD at a premature termination 
codon is not solely based on the absence of Pab1p and suggest that a specific 
recruitment step must commit Upf1p to the process and Upf1p-associated mRNAs to 
NMD.