Spc1 regulates substrate selection for signal peptidase
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Evaluation Summary:
This interesting and well-executed work addresses the function of one of the subunits of the signal peptidase, a complex that processes signal peptides in a wide variety of secretory and membrane proteins. This topic is of relevance to the membrane cell biology community, and the study will be of broader interest when the authors demonstrate the relevance of their findings to the natural substrates of the studied enzyme.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 agreed to share their name with the authors.)
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Abstract
Signal peptidase (SPase) cleaves the signal sequences (SSs) of secretory precursors. It contains an evolutionarily conserved membrane protein subunit, Spc1 that is dispensable for the catalytic activity of SPase, and its role remains elusive. In the present study, we investigated the function of yeast Spc1. First, we set up an in vivo SPase cleavage assay using secretory protein CPY variants with SSs modified in the n and h regions. When comparing the SS cleavage efficiencies of these variants in cells with or without Spc1, we found that signal-anchored sequences become more susceptible to cleavage by SPase without Spc1. Further, SPase-mediated processing of transmembrane (TM) segments in model membrane proteins was reduced upon overexpression of Spc1. Spc1 was co-immunoprecipitated with membrane proteins carrying uncleaved signal-anchored or TM segments. These results suggest a role of Spc1 in shielding TM segments from SPase action, thereby contributing to accurate substrate selection for SPase.
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Reviewer #3 (Public Review):
Signal peptidase is an essential enzyme involved in protein transport and secretion of all organisms. This contribution analyses the function of the non-essential subunit Spc1 of the yeast signal peptidase. The result suggests that Spc1 binds and recognizes hydrophobic membrane anchor sequences that should not be cleaved by the signal peptidase. Numerous variations of the signal peptides from CPY and Sps2 were tested for their cleavage and glycosylation in the presence or absence of Spc1. The authors conclude that the hydrophobicity and length are the main determinants to allow the cleavage.
The provided data are technically perfect and in a good, logical order. However, in contrast to their claim, no real internal membrane anchor sequence was tested. It also remains unclear whether the Sps2 protein has to …
Reviewer #3 (Public Review):
Signal peptidase is an essential enzyme involved in protein transport and secretion of all organisms. This contribution analyses the function of the non-essential subunit Spc1 of the yeast signal peptidase. The result suggests that Spc1 binds and recognizes hydrophobic membrane anchor sequences that should not be cleaved by the signal peptidase. Numerous variations of the signal peptides from CPY and Sps2 were tested for their cleavage and glycosylation in the presence or absence of Spc1. The authors conclude that the hydrophobicity and length are the main determinants to allow the cleavage.
The provided data are technically perfect and in a good, logical order. However, in contrast to their claim, no real internal membrane anchor sequence was tested. It also remains unclear whether the Sps2 protein has to remain in the ER as an uncleaved protein.
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Reviewer #2 (Public Review):
Defining the subunits' role in forming the eukaryotic signal peptidase complex is essential to understand the process of protein maturation and selection. This fundamental knowledge can have direct implications in the context of biotechnological protein production. Here, Yim et al. focus on the yeast signal peptidase protein subunit Spc1. Identifying the role of this membrane protein is a difficult task because this small subunit is non-essential. Using yeast strain lacking Spc1, the authors report that particular model signal-anchored proteins become more susceptible to cleavage by signal peptidases, whereas overproduction of Spc1 tends to reduce their maturation.
The work is carefully presented and executed. For example, the authors employ radioactive pulse-chase experiments for precise tracking of the …
Reviewer #2 (Public Review):
Defining the subunits' role in forming the eukaryotic signal peptidase complex is essential to understand the process of protein maturation and selection. This fundamental knowledge can have direct implications in the context of biotechnological protein production. Here, Yim et al. focus on the yeast signal peptidase protein subunit Spc1. Identifying the role of this membrane protein is a difficult task because this small subunit is non-essential. Using yeast strain lacking Spc1, the authors report that particular model signal-anchored proteins become more susceptible to cleavage by signal peptidases, whereas overproduction of Spc1 tends to reduce their maturation.
The work is carefully presented and executed. For example, the authors employ radioactive pulse-chase experiments for precise tracking of the dynamic protein maturation process and series of signal sequence variants in the N-terminal and hydrophobic region to test the contribution limit of Spc1 in the reaction specificity. They also introduce mass spectrometry controls, to show for instance that deletion of Spc1 does not affect the stability of the other subunits in the signal peptidase complex.
A limitation of the work as presented concerns the mechanism of action of Spc1. It is shown that Spc1 does not affect signal peptidase recognition efficiency, yet it is unclear how Spc1 would regulate the activity of the signal peptidase. Some co-immunoprecipitation experiments suggest that membrane proteins carrying un-cleaved signal-anchor can be isolated with Spc1, suggesting some direct interaction with Spc1. Yet, the extent of this analysis (presented in the last Fig. 6) is insufficient as yet to firmly support the conclusion that Spc1 functions to shield transmembrane segments from signal peptidase action.
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Reviewer #1 (Public Review):
The Signal Peptidase Complex (SPC) processes signal peptides in a wide variety of secretory and membrane proteins that are inserted into the ER membrane in eukaryotic cells. How SPC discriminates transmembrane segments of membrane proteins from signal peptides remains elusive. The work of Kim et al shows that the Spc1 subunit of the SPC enzyme is involved in the quality control function of SPC facilitating accurate cleavage of signal peptides and preventing it from cleaving after transmembrane segments. The work is well executed and the experiments are done in triplicate.
The approach that Kim et al use is to investigate signal peptide cleavage of carboxy-peptidase Y (CPY) variants with extended amino-terminal regions of various lengths and with low, intermediate or high hydrophobicity of the H region of the …
Reviewer #1 (Public Review):
The Signal Peptidase Complex (SPC) processes signal peptides in a wide variety of secretory and membrane proteins that are inserted into the ER membrane in eukaryotic cells. How SPC discriminates transmembrane segments of membrane proteins from signal peptides remains elusive. The work of Kim et al shows that the Spc1 subunit of the SPC enzyme is involved in the quality control function of SPC facilitating accurate cleavage of signal peptides and preventing it from cleaving after transmembrane segments. The work is well executed and the experiments are done in triplicate.
The approach that Kim et al use is to investigate signal peptide cleavage of carboxy-peptidase Y (CPY) variants with extended amino-terminal regions of various lengths and with low, intermediate or high hydrophobicity of the H region of the signal sequence. The results show that signal peptide processing by Signal Peptidase Complex (SPC) depends on the n-region and the h-region. Signal peptide cleavage of the internal signal sequences is greater in the Spc1 knock out strain.
In addition, Kim and coworkers study SPC-mediated cleavage after a TM segment of single spanning and a double spanning membrane protein, based on the model membrane protein leader peptidase. Their results (Fig. 4 and Fig. 5) show Spc1 acts as a negative regulator of cleavage of the model single and double spanning membrane proteins when the stretch is quite hydrophobic. Notably, the presence of Spc1 does not completely eliminate SPC cleavage of the artificial membrane proteins. It is not an all or nothing effect, suggesting that the artificial constructs are not faithfully mimicking a membrane protein, which you do not want to be cleaved.
Given this, it remains unclear what would be observed for physiological membrane proteins that are not normally SP processed but do have a potential signal peptide cleavage site immediately at the C-terminal region. The authors model predicts that such proteins should be cleaved by SPC in the absence of Spc1, and, if this were observed, it would conclusively prove that Spc1 plays a fidelity function and contributes to the accuracy of signal peptide cleavage by SPC.
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Evaluation Summary:
This interesting and well-executed work addresses the function of one of the subunits of the signal peptidase, a complex that processes signal peptides in a wide variety of secretory and membrane proteins. This topic is of relevance to the membrane cell biology community, and the study will be of broader interest when the authors demonstrate the relevance of their findings to the natural substrates of the studied enzyme.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 agreed to share their name with the authors.)
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