bioRxiv preprint doi: https://doi.org/10.1101/179564; this version posted August 22, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. RAC-Ssb and integral membrane protein translation Role for Ribosome-Associated Complex and Stress-Seventy subfamily B (RAC-Ssb) in integral membrane protein translation Ligia Acosta-Sampson1, Kristina Döring2,3, Yuping Lin1,† Vivian Y. Yu1, Bernd Bukau2,3, Günter Kramer2,3, and Jamie H. D. Cate1,4,5 From the 1Department of Molecular and Cell Biology, University of California at Berkeley, Berkeley, CA 94720, USA. 2Center for Molecular Biology of Heidelberg University (ZMBH), DKFZ-ZMBH Alliance, Im Neuenheimer Feld 282, Heidelberg D-69120, Germany. 3German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, Heidelberg D-69120, Germany 4Department of Chemistry, University of California at Berkeley, Berkeley, CA 94720, USA. 5Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA. †Present address: Key Laboratory of Systems Microbial Biotechnology, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China. Running title: RAC-Ssb and integral membrane protein translation Correspondence should be addressed to Jamie H.D. Cate (email: [email protected]). Keywords: membrane protein, protein synthesis, chaperone, major facilitator superfamily (MFS), translation control, ribosome associated complex (RAC), stress-seventy subfamily B (Ssb), unfolded protein response (UPR). ABSTRACT mechanisms underlying human disease and protein Targeting of most integral membrane proteins production in biotechnology. to the endoplasmic reticulum is controlled by the signal recognition particle (SRP), which recognizes a hydrophobic signal sequence near the protein N-terminus. Proper folding of these Integral membrane protein open reading proteins is monitored by the unfolded protein frames (ORFs) make up approximately 20-30% of response, and involves protein degradation eukaryotic genomes (1). Membrane protein (MP) pathways to ensure quality control. Here, we biogenesis is a highly coordinated, multi-phase identify a new pathway for quality control of process in which many of the steps are thought to major facilitator superfamily transporters that be kinetically controlled. Early in membrane occurs before the first transmembrane helix–the protein synthesis, the ribosome nascent chain signal sequence recognized by SRP–is made by (MP-RNC) complex is identified by the signal the ribosome. Increased rates of translation recognition particle (SRP) and targeted to the elongation of the N-terminal sequence of these endoplasmic reticulum (ER), for insertion of the integral membrane proteins can divert the nascent MP into the ER membrane through the Sec protein chains to the ribosome-associated complex translocon (2,3). For membrane proteins, (RAC) and Stress-Seventy Subfamily B (Ssb) exposure of the first transmembrane helix (TM1) chaperones. We also show that quality control of at the ribosome peptide exit site serves as the integral membrane proteins by RAC-Ssb couples signal for SRP binding, though SRP can be translation rate to the unfolded protein response, recruited to the ribosome while TM1 is still inside which has implications for understanding the ribosome exit tunnel (4-8). SRP-target binding is also mediated by the nascent-polypeptide 1 bioRxiv preprint doi: https://doi.org/10.1101/179564; this version posted August 22, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. RAC-Ssb and integral membrane protein translation associated complex (NAC), which prevents non- predicts gene expression levels in E. coli and specific binding of SRP to non-secretory Saccharomyces cerevisiae, whose codon usage substrates, and deters antagonistic interactions frequencies are positively correlated with between SRP and N-terminal-modifying enzymes isoaccepting tRNA abundances (20). during scanning of nascent polypeptides (9-11). Subsequently, a wide-ranging scale, the tRNA These early targeting steps are crucial for proper Adaptation Index (tAI), was introduced that better membrane protein insertion into the ER and to described the relationship between tRNA pools prevent proteostatic stress due to the aggregation and codon usage patterns for unicellular and of the highly hydrophobic transmembrane helices multicellular organisms. The tAI integrates the of membrane proteins in the cytosol (5). tRNA gene copy number and Crick’s wobble rules The contributions of kinetics in controlling for codon-anticodon pairing (21). Global tAI has nascent chain targeting has remained unclear. a strong correlation with high expression levels for SRP-mediated attenuation of translation cytosolic proteins, but this correlation is weak for elongation has been proposed to extend the time membrane and secretory proteins in S. cerevisiae window for SRP targeting in eukaryotes and is (15). necessary for efficient translocation in human cells At the protein level, codon usage preferences (12-14). However, a recent study using SRP- also play an important role in regulating mediated selective ribosome profiling (SeRP) in membrane protein function, folding and structural yeast did not find evidence for general SRP- stability. Several pathogenic synonymous mediated pausing on secretory nascent chain polymorphisms in the human MDR1 gene, which substrates (7). Instead, computational analyses of encodes the P-glycoprotein drug efflux transporter, mRNAs encoding membrane proteins and secreted have been identified that alter the structure and proteins have identified clusters of rare codons efflux activity of the transporter (22,23). that modulate their translation elongation rates to Misfolding and aggregation of secreted and facilitate constructive interactions with SRP membrane proteins trigger the ER quality control (15,16). Tuller and colleagues found that system characterized by two separate processes, transcripts from several Gene Ontology (GO) the unfolded protein response (UPR), a signaling categories, including those composed of secreted pathway that monitors ER homeostasis and and membrane proteins, had a 5’-end slow responds to problems during membrane protein translational ramp of 30-50 codons comprised of biogenesis at the ER, and the ER-associated non-optimal codons that optimizes ribosome degradation pathway (ERAD), which targets spacing and reduces the chances of ribosomal misfolded proteins in the ER lumen and membrane ‘traffic jams’ (17). Moreover, a genome-based for proteasomal degradation (24-26). analysis of N-terminal signal-sequences in Sec- Among the different classes of MPs, the major dependent secreted proteins from E. coli showed facilitator superfamily (MFS) is a ubiquitous that these sequences had a high frequency of non- family of secondary transporters that employ optimal codons (18). Downstream from the N- electrochemical gradients to drive substrate terminus, Pechmann and colleagues showed translocation through the membrane [Transporter through in silico sequence analysis and ribosome Classification Database (TCDB) #2.A.1; (27)] (28). profiling data that secreted and membrane proteins It is the largest family of secondary transporters had REST (mRNA-encoded slowdown of including up to 25% of membrane transporters in translation) elements of non-optimal codons 30-40 prokaryotes (29). Most members range in size codons downstream of the signal-sequence/TM1 from 400 to 600 amino acids and are organized that assisted SRP interactions through slowing into 12 transmembrane α-helices. MFS translation elongation (16). All of these studies transporters are important drug and therapeutic relied on computational methods to discern targets because they transport a variety of patterns of codon usage bias. The earliest metric substrates including simple carbohydrates, designed to detect this bias was the Codon peptides, oligosaccharides and lipids (30). These Adaptation Index (CAI), which is based on the transporters can be divided based on their use of codon usage frequencies of highly expressed genes three distinct transport mechanisms: uniporters, in a given species (19). The CAI adequately symporters and antiporters (28). The sugar porter 2 bioRxiv preprint doi: https://doi.org/10.1101/179564; this version posted August 22, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. RAC-Ssb and integral membrane protein translation subfamily is one of the largest and most widely frequently in yeast (Figure 1B). Other codon studied subfamilies of MFS transporters, and usage metrics such as the codon adaptation index includes the human SCLC2 (GLUT) family of (CAI) and the tRNA adaptation index (tAI) membrane transporters, the HXT family of hexose yielded similar results (Figure S1)(19,21,39). transporters from S. cerevisiae, and the We therefore optimized the codons in the cdt- oligosaccharide family of transporters in 1 sequence to better match the codon preferences Neurospora crassa. The transcriptional regulation of S. cerevisiae. After codon optimization of cdt-1, of these transporters has been extensively studied the CAI metric showed that the optimized version in relation to extracellular substrate concentrations consists of the most