Proteomic mapping of cytosol-facing outer mitochondrial and ER membranes in living human cells by proximity biotinylation

Thank you for submitting your article "Proteomic mapping of outer mitochondrial and ER membranes by proximity biotinylation and discovery of a tethering complex" for consideration by eLife. Your article has been favorably evaluated by Anna Akhmanova (Senior Editor) and three reviewers, one of whom, David Pagliarini (Reviewer #1), is a member of our Board of Reviewing Editors.

The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission. Although we recognize that you have submitted this manuscript as a Tools and Resources paper, the consensus view is that your conclusions require some functional validation as detailed below.

Summary:

This manuscript by Hung et al. describes the use of a previously developed technique, proximity biotinylation, to discover novel proteins localized to cellular compartments with relative inaccessibility to standard experimental techniques including the outer mitochondrial membrane (OMM) and the endoplasmic reticulum membrane (ERM). Through purification of mitochondria from various tissues followed by mass spectrometry-based proteomics and computational approaches, and via previous APEX2-based studies by these authors, much of the mitochondrial proteome has been established and recorded in databases such as MitoCarta2.0. However, probing cytosol-facing proteins of the OMM and ERM presents a challenge due to the loss or depletion of outer membrane proteins during the purification process. Another issue lies in the poor overlap observed between proteomic MS datasets for ER extracts. Proximity biotinylation relies on probing cells using an engineered APEX2 peroxidase genetically modified to target a specific cellular region, a biotin-phenol (BP) substrate, and hydrogen peroxidase (H2O2). Upon the addition of H2O2 for 1 minute, APEX2 converts BP to BP radicals which can then covalently tag proteins in their immediate vicinity. Cells are lysed and the biotinylated proteins are enriched using streptavidin beads followed by MS analysis.

In the present manuscript, the authors construct modified APEX2 peroxidases with targeting signals for native OMM and ERM proteins, as well as a cytosolic control APEX2 containing only a nuclear export sequence. Localization of the constructs was verified via fluorescence microscopy. The authors used Stable Isotope Labelling with Amino acids in Cell culture (SILAC) to radiometrically quantify the relative proximity of each detected protein to their targeted location versus the cytosol. The experiments resulted in the detection of 137 potential OMM proteins, and 634 potential ERM proteins for a total of 94 potentially novel proteins between both locations. The authors chose to focus on closer inspection of the proteins involved in mitochondria-ER contact sites and found SYNJ2BP, a protein of unknown function, was enriched in both the OMM and ER proteomes. Overexpression of SYNJ2BP in HEK 293T cells resulted in increased mitochondrial contacts with the rough endoplasmic reticulum. To uncover the binding partner of SYNBP1, the authors utilized the CRISPR/Cas9 system to remove endogenous SYNBP1 and expressed V5-tagged SYNBP1 in Hela cells followed by immunoprecipitation with anti-V5 beads. The interacting proteins were analyzed through LC-MS/MS. Of the 56 discovered proteins, the authors reasoned that only RRBP1 contained the characteristics necessary to act as the ER binding partner for SYNBP1, presenting a potentially novel mammalian tethering complex between the mitochondria and ER.

Essential revisions:

The reviewers were all in agreement that this was elegant, technically sound investigation into a timely and important area of organellar communication. The reviewers commented on the particularly impressive specificity of the authors' approach, and the use of relevant controls for contact site enriched proteins. The narrative style, including extensive open discussion of the experimental limitations of the work, was also well received. Nonetheless, the reviewers agreed that the further evidence of direct interaction between SYNJ2BP and RRBP1 would greatly strengthen their conclusions, as would some evidence of the functional relevance of the tethering complex. In particular, the reviewers request that the authors address the following concerns.

1) While the overexpression data and localization of SYNJ2BP is convincing, the functional role of the protein within membrane contact sites remains underexplored, as demonstrated by the lack of any observed phenotype for CRISPR KO of SYNJ2BP or RRBP1 (as discussed by the authors). Does a phenotype exist in different cell lines having higher ER-OMM contact density as the authors suggest? In yeast, ERMES is important for mitochondrial lipid biosynthesis. Are there any changes in the lipidomes (whole cell or mitochondrial) of the KO lines? Is mitochondrial phosphatidylserine biosynthesis disrupted? Do any protein abundance or PTM changes exist in the KO lines that indicate a defect in mitochondrial or ER biology? Any changes in mitochondrial biogenesis or OxPhos activity? Of course, all of this issues would not need to be resolved, but some connection to a relevant phenotype would strengthen the authors' central thesis.

2) In their IP-MS experiments, the authors identified 56 interactors for SYNJ2BP. Given the presences of a PDZ domain on this protein, they then searched these 56 for proteins for targets possessing PDZ-binding motifs that might serve as a tethering partner for SYNJ2BP. This analysis led them to RRBP1, the only protein with such a motif. Given the central importance of this PDZ-PDZ-binding motif interaction (the reason why RRBP1 was chosen), the authors should test its importance for the SYNJ2BP overexpression phenotype. This should be doable given that the authors seem to have some of the cloning tools already in hand. How does a SYNJ2BP protein behave with a truncated PDZ domain? Is it possible to use a mapping approach to identify the interaction site between both putative tethers?