Discussion

BioID has been used to profile the proteome of different cellular compartments and molecular complex systems (Varnaitė & MacNeill, 2016). However, this is the first time that BioID has been used to identify proteome of the secretory pathway during the recombinant protein expression. Numerous PPIs guiding the folding, modifications, and trafficking of the secreted and membrane proteins through the secretory pathway are transient (Nyfeler, Michnick, & Hauri, 2005; Schreiber, Haran, & Zhou, 2009) and therefore cannot be captured by conventional methods such as co-immunoprecipitation. Consistent with previous studies (Sears, May, & Roux, 2019), these results showed the BioID can detect weak and transient interactions in situ, and therefore it is a powerful approach to study luminal processes involved in protein secretion. We found that disulfide bridge formation enzymes showed the most significant association with bait proteins enriched in disulfide bonds, supporting their critical roles in protein secretion and maintaining ER stability. A previous study on difficult to express (DTE) monoclonal antibodies showed less recognition by PDI impairs disulfide bridge formation within the antibody light chain (LC) which can initiates the intracellular degradation by the ubiquitin proteasome system via ERAD (Mathias et al., 2020). Thus, insufficient interaction between the secreted proteins with enriched disulfide bond and PDIs can limit secretion efficiency and serve as a rate-limiting step for protein production. In another study, the tissue specific analysis of SecMs expression showed a positive correlation between the expression of P4HB and PDIA4 and liver tissue where numerous disulfide bond enriched proteins are secreted by hepatocytes (Feizi et al., 2017). These observations are clear evidence that suggests the tissue-specific fine-tuning of the PDI family expression level in response to the enrichment of the disulfide sites. Together, these results showed PDIs are actively involved in adaptive responses and secretion of proteins with dominant disulfide bonds which are crucial for restoring ER stability, and therefore yielding the recombinant proteins. Given the associations between the SecMs and the features of the model proteins, we also hypothesized that SecMs preferentially interacting with bait-BirA proteins that carry certain structural features may be essential for the secretion of those proteins. While evidence linking SecMs to the structural motifs is lacking, many molecular chaperones selectively interact with certain sequence and structural elements to favor the particular folding pathways (Gidalevitz, Stevens, & Argon, 2013). For example, chaperones of the HSP70 family evolved to bind extended β strand peptides; interestingly, the associations identified between chaperones and asx motif and ST turn represent a novel association for further study.
While we show BioID works well for studying the synthesis of secreted proteins, we acknowledge that biotin-based methods have some limitations as well. Biotin is actively imported into the cytoplasm of cells and can freely diffuse to the nucleus, but it may not be as accessible in the secretory pathway, thus reducing labeling efficacy in that compartment (Kim & Roux, 2016). Here we showed this challenge is not an insurmountable issue, in that the BioID2 construct successfully identified many expected luminal interactions. BioID2 requires less biotin supplementation, and exhibits enhanced labeling of proximate protein (Kim et al., 2016) allowing for BioID to be introduced to new systems where biotinylation supplementation may not be easily accomplished (Sears et al., 2019). More recently, two promiscuous mutants of biotin ligase, TurboID and miniTurbo, have been developed to catalyze proximity labeling even with much greater efficiency (Branon et al., 2020) and therefore can be considered as an effective method when proximal labeling of the endomembrane organelles is desired.
In summary, we demonstrate here an approach to identify the protein interactions that synthesize and support secreted proteins, and thus define the product-specific secretory pathway. The identification of such machinery opens avenues for mammalian synthetic biology, wherein biotherapeutic production hosts can be rationally engineered to improve the titer and quality of diverse proteins in a client specific manner.