A systematic proximity ligation approach to studying protein‐substrate specificity identifies the substrate spectrum of the Ssh1 translocon

Abstract

Many cellular functions are carried out by protein pairs or families, providing robustness alongside functional diversity. For such processes, it remains a challenge to map the degree of specificity versus promiscuity. Protein–protein interactions (PPIs) can be used to inform on these matters as they highlight cellular locals, regulation and, in cases where proteins affect other proteins ‐ substrate range. However, methods to systematically study transient PPIs are underutilized. In this study, we create a novel approach to systematically compare stable or transient PPIs between two yeast proteins. Our approach, Cel‐lctiv (CELlular biotin‐Ligation for Capturing Transient Interactions in vivo ), uses high‐throughput pairwise proximity biotin ligation for comparing PPIs systematically and in vivo . As a proof of concept, we studied the homologous translocation pores Sec61 and Ssh1. We show how Cel‐lctiv can uncover the unique substrate range for each translocon allowing us to pinpoint a specificity determinator driving interaction preference. More generally, this demonstrates how Cel‐lctiv can provide direct information on substrate specificity even for highly homologous proteins. image Protein‐protein interactions (PPIs) can reveal information about cellular localization, regulation, and substrate range of protein pairs or families. A new biotin ligation approach allows systematic study and comparison of stable and transient PPIs in vivo . The Cel‐lctiv (CELlular biotin‐Ligation for Capturing Transient Interactions in vivo ) method systematically compares stable or transient PPIs between yeast proteins. Cel‐lctiv uses high‐throughput, pairwise proximity‐biotin ligation in vivo . Proof‐of‐concept application of Cel‐lctiv to the homologous translocation pores Sec61 and Ssh1 defines their unique substrate ranges. Cel‐lctiv uncovers a unique signal peptide feature that differentiates Sec61 and Ssh1 substrates.