A sequence resembling a peroxisomal targeting sequence directs the interaction between the tetratricopeptide repeats of Ssn6 and the homeodomain of α2

Abstract

The tetratricopeptide repeat (TPR) is a 34-aa sequence motif, typically found in tandem clusters, that occurs in proteins of bacteria, archea, and eukaryotes. TPRs interact with other proteins, although few details on TPR-protein interactions are known. In this paper we show that a portion of a loop in the homeodomain of the DNA-binding protein ␣2 is required for its recognition by the TPRs of the corepressor Ssn6. The amino acid sequence of this loop is similar to the sequences recognized by the TPRs of an entirely different protein, Pex5, which directs peroxisomal import. We further show that ␣2 can be made to bind specifically in vitro to the TPRs of Pex5 and that a point mutation that disrupts the ␣2-Ssn6 interaction also disrupts the ␣2-Pex5 interaction. These results demonstrate that two different TPR proteins recognize their target by a similar mechanism, raising the possibility that other TPRtarget interactions could occur through the same means.

The Short-Lived Matα2 Transcriptional Repressor Is Protected from Degradation In Vivo by Interactions with Its Corepressors Tup1 and Ssn6

“…This conclusion is based on (i) the demonstration that the binding of Tup1 to the N terminus of ␣2 is required for Tup1 to block the ubiquitination and turnover of proteins containing the Deg1 signal from ␣2 (Fig. 4 and 5), (ii) the finding that Tup1 and Ssn6 are associated together in a protein complex (39,44), and (iii) the observation, noted above, that Ssn6 binds directly to the homeodomain in the C-terminal domain of ␣2 (34,36). Furthermore, we have observed that the stabilization of the ␣2 protein by TUP1 and SSN6 overexpression is abolished by mutations that impair ␣2-corepressor binding (Fig.…”

“…6C and D). These experiments utilized a mutant form of ␣2 containing the L10S and R173A amino acid substitutions; these single mutations have been isolated previously and shown to disrupt the ␣2-Tup1 and ␣2-Ssn6 interactions, respectively (22,34). The ␣2 L10S/R173A double mutant was expressed in cells, and the degradation of this protein was examined by pulse-chase assays.…”

“…The binding of these two proteins to sequences in the upstream region of a-specific genes tethers the Tup1-Ssn6 general repression complex in the vicinity of a-specific gene promoters (19,20), where it potently represses transcription by a variety of mechanisms (35). This repression complex is recruited to target promoters by ␣2 through several distinct protein-protein interactions: Tup1 binds to the N-terminal domain of ␣2, while Ssn6 directly contacts the homeodomain in the C terminus of the protein (22,34,36).…”

“…The binding of these two proteins to sequences in the upstream region of a-specific genes tethers the Tup1-Ssn6 general repression complex in the vicinity of a-specific gene promoters (19,20), where it potently represses transcription by a variety of mechanisms (35). This repression complex is recruited to target promoters by ␣2 through several distinct protein-protein interactions: Tup1 binds to the N-terminal domain of ␣2, while Ssn6 directly contacts the homeodomain in the C terminus of the protein (22,34,36).Although ␣2 directs the extremely robust and stable repression of a-specific genes in ␣ haploid cells, the ␣2 protein itself is very short lived in vivo (half-life, Ͻ5 min) (16). This rapid degradation is carried out by the ubiquitin-proteasome system (4, 14), which plays an essential role in a wide array of diverse cellular processes (9,13,43).…”

Identification of peroxisomal targeting signal of pumpkin catalase and the binding analysis with PTS1 receptor

“…Pex5p, a 64-69 kDa protein, has TPR (tetratricopeptide repeat) motifs in the Cterminal part and is localized in the cytosol. The TPR motif that consists of 34 amino acids is necessary and sufficient for the binding of Pex5p (Brocard et al, 1994;Smith and Johnson, 2000;Terlecky et al, 1995). On the other hand, PEX5 genes in plants and humans have been cloned based on the sequence of yeast PEX5 gene (Brickner et al, 1998;Kragler et al, 1998;Wiemer et al, 1995;Wimmer et al, 1998).…”