Protein Moonlighting Revealed by Noncatalytic Phenotypes of Yeast Enzymes

Abstract

A single gene can partake in several biological processes, and therefore gene deletions can lead to different-sometimes unexpected-phenotypes. However, it is not always clear whether such pleiotropy reflects the loss of a unique molecular activity involved in different processes or the loss of a multifunctional protein. Here, using metabolism as a model, we systematically test the null hypothesis that enzyme phenotypes depend on a single annotated molecular function, namely their catalysis. We screened a set of carefully selected genes by quantifying the contribution of catalysis to gene deletion phenotypes under different environmental conditions. While most phenotypes were explained by loss of catalysis, slow growth was readily rescued by a catalytically inactive protein in about one-third of the enzymes tested. Such noncatalytic phenotypes were frequent in the Alt1 and Bat2 transaminases and in the isoleucine/valine biosynthetic enzymes Ilv1 and Ilv2, suggesting novel "moonlighting" activities in these proteins. Furthermore, differential genetic interaction profiles of gene deletion and catalytic mutants indicated that is functionally associated with regulatory processes, specifically to chromatin modification. Our systematic study shows that gene loss phenotypes and their genetic interactions are frequently not driven by the loss of an annotated catalytic function, underscoring the moonlighting nature of cellular metabolism.

Understanding protein multifunctionality: from short linear motifs to cellular functions

“…An illustrative example is the recent study of Espinosa-Cantú and colleagues, in which they experimentally addressed the prevalence of enzymes with moonlighting functions in Saccharomyces cerevisiae [12]. They evaluated if the enzyme gene deletion phenotypes are caused solely by the loss of catalytic activity, or to a yet unknown moonlighting function independent of the catalytic activity [12]. This study showed that 4 out of 11 tested enzymes may have moonlighting functions, thus suggesting that moonlighting proteins may be highly prevalent.…”

Multiple Forms of Multifunctional Proteins in Health and Disease

“…The molecular activities of a moonlighting protein are usually described as unrelated to each other and independently of the primary sequence (Huberts and van der Klei, 2010;Flores and Gancedo, 2011). Indeed, experimental evidence for the characterization of a moonlighting protein is mostly provided by mutants that are defective in one molecular function without affecting the others (Jeffery, 2009;Gancedo et al, 2016;Espinosa-Cantu et al, 2018). In the case of yeast Ure2, mutants without nitrogen-catabolite repression activity maintain their enzymatic capacities ( Figure 1D).…”

“…Moonlighting proteins with different molecular and functional characteristics have been described in a wide variety of organisms and the number of examples has increased in the last few years to almost 700 carefully curated moonlighting proteins (Hernández et al, 2014;Mani et al, 2015;Chen et al, 2018;Franco-Serrano et al, 2018b). Even though experimental setups have been aimed at revealing protein moonlighting in an unbiased manner (Su and Pillus, 2016;Espinosa-Cantu et al, 2018), still most known cases have been described by serendipity, either by studying a single protein in an exhaustive manner (e.g., the delta-crystalline Piatigorsky and Horwitz, 1996) or as rather surprising results from functional screens (Zelenaya-Troitskaya et al, 1995;Hall et al, 2004;Chen et al, 2005;Scott and Pillus, 2010;Scherrer et al, 2010;Chang et al, 2012).…”

“…Huge amounts of data are being produced from experimental and computational analysis in functional genomics and systems biology, making it urgent to unify definitions of protein multifunctionality in a more systematic manner. Recent advances in large-scale genetic and genetic-interaction screens (Bellay et al, 2011;Martin et al, 2015;Espinosa-Cantu et al, 2018), proteomics and protein-protein interaction databases (Gomez et al, 2011;Becker et al, 2012;Chapple et al, 2015;Wang and Jeffery, 2016), and metabolite screens (Sevin et al, 2017;Piazza et al, 2018), along with computational analyses of functional data (Ekman et al, 2005;Nam et al, 2012;Pritykin et al, 2015) have huge potential to reveal patterns of protein multifunctionality in living systems.…”

“…In addition, multifunctional proteins may coordinate the crosstalk of dissimilar biological processes, which is specially relevant in organisms with large genomes and intricate metabolic and regulatory pathways (Flores and Gancedo, 2011;Irving et al, 2018). Thus, the presence of undetected multifunctionality is a confounding factor in functional association analyses based on guilt-by-association, genotype-phenotype maps, and homology prediction and annotation (Jeffery, 1999;Salathe et al, 2006;Gillis and Pavlidis, 2011;Espinosa-Cantu et al, 2018). Identification and understanding of protein multifunctionality is needed to grant a better understanding of living systems in health and disease.…”