Intracellular quality control mechanisms prevent misregulation and cytotoxicity. Protein degradation systems maintain protein quality by destroying potentially harmful incomplete or faulty polypeptides. Unfinished protein fragments are often the products of ribosomes that stall on mRNA transcripts. Ribosomal stalling also prevents normal termination and mRNA dissociation, thereby depleting the pool of ribosomes available for translation. In bacteria, a mechanism known as tmRNA rescue frees stalled ribosomes and marks incomplete proteins for destruction. Although this system has been studied in depth within the context of cytosolic proteins, relatively little is known about its effectiveness towards membrane proteins. Given that a significant portion of genomes of both simple and complex organisms encode membrane proteins, it is important to understand how stalling events of this type are handled and how protein quality control mechanisms operate in the challenging environment of the membrane interface. The overall goal of this project is to study the physiological response to ribosomes stalled during the synthesis of membrane proteins. Experiments conducted directly in bacteria will seek to understand the role of the tmRNA system in labeling incomplete membrane proteins for destruction as well as the role of specific proteases in degrading these proteins. The effect of membrane protein topology on degradation will also be ascertained. Next, quantitative proteomic methods will be used to determine the extent of endogenous membrane protein labeling by the tmRNA system and which of these proteins are degraded by proteases. Finally, an in vitro system will be developed to examine mechanistically the degradation of membrane proteins by different ATP-dependent proteases. It is expected that these experiments will yield a more comprehensive view of protein quality control in cells. A better understanding of protein quality control in bacteria could lead o the discovery of new targets for therapeutic development to combat bacterial infections.

Public Health Relevance

Regulated protein degradation mechanisms are used in cells to maintain protein quality. The goal of this project is to gain a deeper understanding of quality control of membrane proteins in bacteria. Insight into bacterial mechanisms of quality control is important for human health because it may lead to the discovery of new therapeutic targets to fight bacterial infection.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Postdoctoral Individual National Research Service Award (F32)
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Special Emphasis Panel (ZRG1)
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Lees, Robert G
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Massachusetts Institute of Technology
Schools of Arts and Sciences
United States
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Hari, Sanjay B; Grant, Robert A; Sauer, Robert T (2018) Structural and Functional Analysis of E. coli Cyclopropane Fatty Acid Synthase. Structure 26:1251-1258.e3
Amberg-Johnson, Katherine; Hari, Sanjay B; Ganesan, Suresh M et al. (2017) Small molecule inhibition of apicomplexan FtsH1 disrupts plastid biogenesis in human pathogens. Elife 6:
Hari, Sanjay B; Sauer, Robert T (2016) The AAA+ FtsH Protease Degrades an ssrA-Tagged Model Protein in the Inner Membrane of Escherichia coli. Biochemistry 55:5649-5652