Gazi, Z. | Search bioRxiv

bioRxiv2022

A toolbox for systematic discovery of stable and transient protein interactors in baker's yeast

Identification of both stable and transient interactions is essential for understanding protein function and regulation. While assessing stable interactions is more straightforward, capturing transient ones is challenging. In recent years, sophisticated tools have emerged to improve transient interactor discovery, with many harnessing the power of evolved biotin ligases for proximity labelling. However, biotinylation-based methods have lagged behind in the model eukaryote, Saccharomyces cerevisiae, possibly due to the presence of several abundant, endogenously biotinylated proteins. In this study, we optimised robust biotin- ligation methodologies in yeast and increased their sensitivity by creating a bespoke technique for downregulating endogenous biotinylation which we term ABOLISH (Auxin- induced BiOtin LIgase diminiSHing). We used the endoplasmic reticulum insertase complex (EMC) to demonstrate our approaches and uncover new substrates. To make these tools available for systematic probing of both stable and transient interactions, we generated five full-genome collections of strains in which every yeast protein is tagged with each of the tested biotinylation machineries; some on the background of the ABOLISH system. This comprehensive toolkit enables functional interactomics of the entire yeast proteome.

molecular biology↗

bioRxiv2022

Determining the targeting specificity of the selective peroxisomal targeting factor Pex9

Targeting proteins to their correct cellular location is a fundamental process that allows them to carry out their cellular functions. Peroxisomes utilize two paralog targeting factors, Pex5 and Pex9, for proteins with a Peroxisomal Targeting Signal 1 (PTS1). However, in spite of their similarity, Pex9 targets only a subset of Pex5 cargo proteins. Here, we studied the properties that facilitate the targeting specificity of Pex9, both by unbiased screens and by site-directed mutagenesis of the PTS1 motifs of either binders or non-binders. We find that the binding specificity of Pex9 is largely determined by the hydrophobic nature of the amino acid preceding the PTS1 tripeptide of its cargos. This is in line with structural modeling of the PTS1-binding cavity of the two factors, showing that while Pex5 has large negative electrostatic patches at the area surrounding the PTS1 binding cavity, Pex9 is mostly hydrophobic. Our work outlines the mechanism by which targeting specificity is achieved, enabling dynamic rewiring of the peroxisomal proteome in changing metabolic needs.

cell biology↗