A conserved translation factor is required for optimal synthesis of a membrane protein family in mycobacteria

Abstract

14Ribosomes require the activity of associated GTPases to synthesize proteins. Despite strong 15 evolutionary conservation, the roles of many of these remain unknown. For example, LepA (also known 16 as elongation factor 4) is a ribosome-associated GTPase found in bacteria, mitochondria, and 17 chloroplasts, yet its physiological contribution to cell survival is not clear. Recently, we found that loss of 18 lepA in Mycobacterium smegmatis (Msm) altered tolerance to rifampin, a drug that targets a non-19 ribosomal cellular process. To uncover the determinants of LepA-mediated drug tolerance, we 20 characterized the whole-cell proteomes and transcriptomes of a lepA deletion mutant relative to a wild-21 type strain. We find that LepA is important for the steady-state abundance of an outer membrane porin, 22which is integral to nutrient uptake and drug susceptibility. Loss of LepA leads to a decreased amount 23 of porin in the membrane, resulting in the drug tolerance phenotype of the lepA mutant. LepA control 24 requires a sequence motif in the 5' region of the porin transcript. Thus, LepA controls the abundance of 25 specific proteins, likely through its activity during translation. 27 Importance 28Our understanding of how ribosomes properly synthesis an entire cellular proteome, in all its 29 complexity, is still evolving. Ribosomal GTPases are often highly conserved, but the roles of many are 30 not well understood. For example, elongation factor 4, or LepA, is a ribosome-associated GTPase 31 conserved across bacteria, mitochondria, and chloroplasts. Using whole-cell proteomics and RNA-32 sequencing of wild type and a lepA deletion mutant, we find that LepA improves translation of 33 mycobacterial porins in a message-specific manner. As porins play a key role in cell wall permeability, 34 loss of LepA produces a plethora of phenotypic changes. These findings underline the problem of 35 building proteins into a complex cell wall, such as that of mycobacteria, and point to a solution in the 36 use of GTPases such as LepA, that have evolved to aid in specific protein synthesis. 37 38 Keywords 39 LepA; ribosome; mycobacteria; porin; drug susceptibility 40 41 45 to adaptive, cellular responses. Indeed, the ribosome machinery and its associated factors/RNA 46 species adapt the cell during periods of environmental transition [2] [3, 4]. Recent advances in 47 techniques such as quantitative proteomics, ribosome profiling, and cryo-electron microscopy (cryo-48 EM) have revealed an intricate balance of mRNA sequences and associating proteins at the ribosome 49 that are necessary for the homeostatic cellular proteome [5-7]. Regardless of growth environment,50bacterial cells are likely composed of a heterogenous population of ribosomes [8, 9], with some inactive 51 and others actively translating parts of the proteome [10, 11]. Associating factors and RNA species tune 52 each ribosome, creating a cell with a spatially-and temporally-regulated proteome [8, 12]. 53The successful synthesis of a given protein at the ribosome is ...

Clinically relevant mutations in mycobacterial LepA cause rifampicin-specific phenotypic resistance

“…LepA was also identified in another forward genetic screen that specifically investigated intra-cellular concentrations of the fluorescent dye calcein, which was in turn associated with rifampicin tolerance 14 . A recent preprint implicates LepA in the synthesis of a mycobacterial porin, and in turn rifampicin and vancomycin tolerance 30 . However, both our data and that of Rubin et al suggests that lepA deletion has potentially wider-ranging perturbations of transcriptional and translational responses.…”