A systematic approach to study protein-substrate specificity enables the identification of Ssh1 substrate range

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 study transient PPIs systematically are underutilized. In this study we create a novel approach to study stable as well as transient PPIs in yeast. Our approach, Cel-lctiv ( <underline>CEL</underline> lular biotin- <underline>L</underline> igation for <underline>C</underline> apturing <underline>T</underline> ransient I nteractions in <underline>V</underline> ivo ), uses high- throughput pairwise proximity biotin ligation for uncovering PPIs systematically and in vivo . As a proof of concept we study 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.