Reference: Harty C and Romisch K (2013) Analysis of Sec61p and Ssh1p interactions in the ER membrane using the split-ubiquitin system. BMC Cell Biol 14:14

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Abstract


BACKGROUND: 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. 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. RESULTS: We 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 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. CONCLUSIONS: We 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.

Reference Type
Journal Article | Research Support, Non-U.S. Gov't
Authors
Harty C, Romisch K
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