Analysis of Sec61p and Ssh1p interactions in the ER membrane using the split-ubiquitin system
Bibliographic record
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 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. 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.
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How this classification was reachedexpand
Full frame distilled prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. Learned from the 10,348 direct Codex labels and 10,348 direct Gemma labels. Candidate is the union of thresholded teacher heads; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels or direct frontier model labels.
Codex and Gemma teacher scores by category
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.000 |
| Insufficient payload (model declined to judge) | 0.000 | 0.000 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one teacher head, not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".