Abstract
BackgroundThe split-ubiquitin system monitors interactions of transmembrane proteins in yeast. It is based on the formation of a quasi-native ubiquitin structure upon interaction of two proteins to which the N- and C-terminal halves of ubiquitin have been fused. In the system we use here ubiquitin formation leads to proteolytic cleavage liberating a transcription factor (PLV) from the C-ubiquitin (C) fusion protein which can then activate reporter genes. Generation of fusion proteins is, however, rife with problems, and particularly in transmembrane proteins often disturbs topology, structure and function.ResultsWe show that both the Sec61 protein which forms the principal protein translocation channel in the endoplasmic reticulum (ER) membrane, and its non-essential homologue, Ssh1p, when fused C-terminally to CPLV are inactive. In a heterozygous diploid Sec61-CPLV is present in protein translocation channels in the ER membrane without disturbing their function and displays a limited set of protein-protein interactions similar to those found for the wildtype protein using biochemical methods. Although its expression level is similar, Ssh1-CPLV interactions are less strong, and, in contrast to Sec61p, Ssh1p does not distinguish between Sbh1p and Sbh2p. We show that interactions can be monitored by reporter gene activity or directly by PLV cleavage, which is more sensitive, but leads to quantitatively different results.ConclusionsWe conclude that the split-ubiquitin system we used here has high fidelity, but low sensitivity and is of limited use for detection of new, transient interactions with protein translocation channels in the ER membrane.
Highlights
The split-ubiquitin system monitors interactions of transmembrane proteins in yeast
Sec61-CPLV and Ssh1-CPLV are dysfunctional In order to generate a strain in which interactions with Sec61p could be monitored using the split-ubiquitin system, we initially tried to integrate the SEC61-CPLV construct (Figure 2A) into the chromosomal SEC61 locus of the haploid reporter strain L40 [10]
Since there are no antibodies against Ssh1p, we assessed expression of the fusion protein in the SSH1/SSH1-CPLV strain by comparing it to the amount of Sec61-CPLV in the heterozygous SEC61/ SEC61-CPLV strain using an antibody against CPLV that we had raised and against Pdi1p as an internal marker, We found that expression levels of both fusion proteins were comparable (Figure 3A, right)
Summary
The split-ubiquitin system monitors interactions of transmembrane proteins in yeast. It is based on the formation of a quasi-native ubiquitin structure upon interaction of two proteins to which the N- and C-terminal halves of ubiquitin have been fused. Sec61p is the core component of the protein translocation channel in the ER membrane, and its association with other proteins determines whether it functions in cotranslational or posttranslational secretory protein transport into the ER [1]. Sec61p and two small, tail-anchored proteins, Sbh1p and Sss1p, form channels for cotranslational protein import into the ER [1]. Ssh1p forms channels with the homologue of Sbh1p, a protein called Sbh2p, and Sss1p [4]. The heptameric Sec complex mediates posttranslational protein import into the yeast ER [1]. A fraction of yeast Sec channels can be found in large complexes with proteasomes and the Hrd ubiquitin ligase in the ER membrane which are likely engaged in dislocation and degradation of misfolded secretory proteins [2,7]
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