Fig. 7.

Stm1p and Def1p are biotinylated by the Asc1-BirA* fusion protein at several sites. A, At glucose deprivation the ribosomal clamping factor Stm1p is located in between the 40S and 60S subunits and prevents mRNA translation. At this condition, Asc1p forms a tightly connected network with Rps3p and Stm1p at the ribosome. B, Stm1p appears as a protein with almost no intrinsic structure during glucose starvation. Biotinylated lysine residues are indicated by red dots. The crystal structure data derive from the PDB entry 4V88 (10) and were used for visualization with the PyMOL Molecular Graphics System software. C, Def1p was biotinylated mainly in the N-terminal half of the protein. Proteasome-dependent processing of Def1p in response to transcription stress occurs before amino acid 530, and the presence of a C-terminal peptide confirms cytoplasmic proximity of Def1p to Asc1p. Biotinylated lysine residues are colored in red. The area of Def1p-processing is indicated in blue and peptides identified by LC-MS analysis are highlighted in green.