Translation Levels Control Multi-Spanning Membrane Protein Expression 2,3*. Hok Seon Kim1., James A. Ernst , Cecilia 1B,Jroenwnny Bostrom1,2, Germaine Fuh1,2, Chingwei V. Lee1,2, Arthur Huang1, Richard L. Vandlen3, Daniel G. Yansura1,3* 1 Department of Antibody Engineering, Genentech Inc., South San Francisco, California, United States of America, 2 Department of Early Discovery Biochemistry, Genentech Inc., South San Francisco, California, United States of America, 3 Department of Protein Chemistry, Genentech Inc., South San Francisco, California, United States of America Abstract Attempts to express eukaryotic multi-spanning membrane proteins at high-levels have been generally unsuccessful. In order to investigate the cause of this limitation and gain insight into the rate limiting processes involved, we have analyzed the effect of translation levels on the expression of several human membrane proteins in Escherichia coli (E. coli). These results demonstrate that excessive translation initiation rates of membrane proteins cause a block in protein synthesis and ultimately prevent the high-level accumulation of these proteins. Moderate translation rates allow coupling of peptide synthesis and membrane targeting, resulting in a significant increase in protein expression and accumulation over time. The current study evaluates four membrane proteins, CD20 (4-transmembrane (TM) helixes), the G-protein coupled receptors (GPCRs, 7-TMs) RA1c and EG-VEGFR1, and Patched 1 (12-TMs), and demonstrates the critical role of translation initiation rates in the targeting, insertion and folding of integral membrane proteins in the E. coli membrane. Citation: Kim HS, Ernst JA, Brown C, Bostrom J, Fuh G, Lee CV, et al. (2012) Translation Levels Control Multi-Spanning Membrane Protein Expression. PLoS ONE 7(4): e35844. doi:10.1371/journal.pone.0035844 Editor: Tobias Spielmann, Bernhard Nocht Institute for Tropical Medicine, Germany Received June 23, 2011; Accepted March 26, 2012; Published April 26, 2012 Copyright: 2012 Kim et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: These authors have no support or funding to report. Competing Interests: Dr. Kim, Dr. Ernst, Dr. Brown, Dr. Bostrom, Dr. Fuh, Dr. Lee, Dr. Huang, Dr. Vandlen and Dr. Yansura are employees of Genentech Inc. There are no patents, products in development or marketed products to declare. This does not alter the authors’ adherence to all the PLoS ONE policies on sharing data and materials. * E-mail: ernst.james@gene.com (JAE); yansura.dan@gene.com (DGY) . These authors contributed equally to this work. Introduction High-level expression of eukaryotic multi-spanning membrane proteins is particularly difficult in E. coli for unknown reasons. While many eukaryotic proteins can be secreted into the periplasm in significant quantities, it remains unknown what limits the accumulation of these polytopic membrane proteins. Eukaryotic and prokaryotic cells share significant homology in both co-translational and post-translational membrane protein insertion mechanisms [1]. In prokaryotes such as E. coli, the post- translational mechanism is used primarily for secreted periplasmic proteins while a co-translational mechanism is used for integral membrane proteins [2]. However, in the cells of higher eukaryotes, such as mammals, the co-translational system is used almost exclusively for both integral membrane and secreted proteins. Co-translational membrane insertion proceeds through several biochemical steps involving three different protein complexes. Initially, the signal recognition particle (SRP) recognizes and binds the first transmembrane or signal peptide domain as it emerges from the ribosome. A SRP receptor (SR) [1] binds to the SRP and docks the ribosome with the protein-conducting channel of the translocon, which creates a pore for insertion of the emerging polypeptide across the lipid bilayer. The hydrophobicity of a region of 20 to 40 residues in the emerging N-terminal domain of the nascent polypeptide determines the engagement of the SRP, and adjacent charged residues determine the orientation of the peptide in the cell membrane [3]. It has been shown that certain components of the E. coli SRP can be functionally substituted for their eukaryotic homologues [4], emphasizing the similarities of the two systems. The number of SRP complexes in eukaryotes suggests one important difference in protein membrane targeting mechanisms. Eukaryotic cells typically contain approximately 10,000 copies of SRP particles or approximately 1 SRP per 10 ribosomes [5]. By comparison, the prokaryotic SRP is present at much lower copy number, often just a single SRP per 100 to 1,000 ribosomes, or as few as 50 particles per cell. The eukaryotic and prokaryotic SRP also have different regulatory functions. In E. coli, the Ffh-4.5S RNA component of the SRP does not contain a functionally analogous region to the Alu domain of the eukaryotic SRP [6,7] and thus lacks a corresponding translation pause mechanism. Further compounding the regulatory differences between eukaryotes and prokaryotes, translation elongation rates in E. coli cells can exceed the rate in eukaryotic cells by as much as ten fold. All of these factors result in an extremely short time period during which the emerging hydrophobic polypeptide chain in E. coli may interact effectively with the membrane bound translocation machinery, unless some other pause mechanism exists. Several mechanisms have been postulated to explain the problems with membrane protein expression. These rationales include available membrane area and protein crowding in the PLoS ONE | www.plosone.org 1 April 2012 | Volume 7 | Issue 4 | e35844