The question how proteins fold is especially pointed for large multidomain, multispanning membrane proteins with complex topologies. We have uncovered the sequence of events that encompass proper folding of the ABC transporter CFTR in live cells, by combining kinetic radiolabeling with protease-susceptibility assays. We found that CFTR folds in two clearly distinct stages. The first, co-translational, stage involves folding of the 2 transmembrane domains TMD1 and TMD2, plus one nucleotide-binding domain, NBD1. The second stage is a simultaneous, post-translational increase in protease resistance for both TMDs and NBD2 caused by assembly of these domains onto NBD1. Our technology probes every 2-3 residues (on average) in CFTR. This in-depth analysis at amino-acid level allows detailed analysis of domain folding and importantly also the next level: the assembly of the domains to native, folded CFTR. Defects and changes brought about by medicines, chaperones or mutations also are amenable to analysis. We here show that the DXD motif in NBD1 that was identified to be required for export of CFTR from the ER turned out to be required for proper domain folding and assembly instead, upstream of transport. CFTR mutated in this motif phenocopies the misfolding and degradation of the well-known disease-causing mutant F508del that established cystic fibrosis as protein-folding disease. The highly modular process of domain folding and stepwise domain assembly explains the relatively high fidelity of folding and the importance of a step-wise folding process for such complex proteins.

Water temperature is one of the most important factors in fish physiology; thus, it is important to identify genes that respond to changes in water temperature. In this study, we identified a warm- temperature acclimation-associated 65-kDa protein (Wap65) in the Kumgang fat minnow Rhynchocypris kumgangensis, a small, cold-freshwater fish species endemic to Korea. Kumgang fat minnow Wap65-1 (kmWap65-1) was cloned using polymerase chain reaction (PCR)-based strategies, and was found to be highly homologous with teleost Wap65-1 and mammalian hemopexin, a heme-binding protein that transfers plasma heme into hepatocytes. kmWap65-1 mRNA was expressed mainly in the liver and its expression levels were significantly increased by both short- and long-term exposure to high temperature, which was evaluated by real-time quantitative PCR. Furthermore, the expression levels of kmWap65-1 were highly elevated by exposure to bacterial lipopolysaccharide. These results indicate that kmWap65-1 expression is associated with environmental stresses such as increases in water temperature and bacterial infection. J. Exp. Zool. 325A:65-74, 2016. © 2015 Wiley Periodicals, Inc.

The question how proteins fold is especially pointed for large multi-domain, multi-spanning membrane proteins with complex topologies. We have uncovered the sequence of events that encompass proper folding of the ABC transporter CFTR in live cells by combining kinetic radiolabeling with protease-susceptibility assays. We found that CFTR folds in two clearly distinct stages. The first, co-translational, stage involves folding of the 2 transmembrane domains TMD1 and TMD2, plus one nucleotide-binding domain, NBD1. The second stage is a simultaneous, post-translational increase in protease resistance for both TMDs and NBD2, caused by assembly of these domains onto NBD1. Our assays probe every 2–3 residues (on average) in CFTR. This in-depth analysis at amino-acid level allows detailed analysis of domain folding and importantly also the next level: assembly of the domains into native, folded CFTR. Defects and changes brought about by medicines, chaperones, or mutations also are amenable to analysis. We here show that the well-known disease-causing mutation F508del, which established cystic fibrosis as protein-folding disease, caused co-translational misfolding of NBD1 but not TMD1 nor TMD2 in stage 1, leading to absence of stage-2 folding. Corrector drugs rescued stage 2 without rescuing NBD1. Likewise, the DxD motif in NBD1 that was identified to be required for export of CFTR from the ER we found to be required already upstream of export as CFTR mutated in this motif phenocopies F508del CFTR. The highly modular and stepwise folding process of such a large, complex protein explains the relatively high fidelity and correctability of its folding.

Summary Water temperature is one of the most important factors in the survival of aquatic organisms. We investigated the effects of increased water temperature on the physiology of Kumgang fat minnow Rhynchocypris kumgangensis, a small cold-water species of freshwater fish that is endemic to Korea. We examined how organ indices and oocyte histology differed by season and exposure to high water temperatures. Fish were collected from streams in Korea during three different seasons, maintained at the same temperatures at which they were collected for three weeks, and then exposed to either control temperatures reflecting the same conditions from which they were collected (15.0 C, August; 7.8 C, November; and 12.3 C, April) or experimental temperatures that were increased a total of 7 C over seven days (to 22.0 C, August; 14.8 C, November; and 19.3 C, April) and maintained at the final temperature for an additional three days. Among the studied organ indices, the gonadosomatic indexes (GSI) in both sexes differed by season, and were significantly decreased by exposure to increased water temperatures during the germ-cell-developing season. The histological features of oocytes also differed by season, and exposure to increased water temperatures during the relevant season markedly attenuated oocyte development. These findings highlight sensitivity of both GSI and oocyte histology on water temperature change, and the possible negative effects of high water temperature on reproduction in Kumgang fat minnow.

N-linked glycosylation of proteins entering the secretory pathway is an essential modification required for protein stability and function. Previously, it has been shown that there is a temporal relationship between protein folding and glycosylation, which influences the occupancy of specific glycosylation sites. Here, we used an in vitro translation system that reproduces the initial stages of secretory protein translocation, folding and glycosylation under defined redox conditions. We found that the efficiency of glycosylation of hemopexin was dependent upon a robust NADPH-dependent cytosolic reductive pathway, which could be mimicked by the addition of a membrane-impermeable reducing agent. We identified a hypoglycosylated acceptor site that is adjacent to a cysteine involved in a short-range disulfide. We show that efficient glycosylation at this site is influenced by the cytosolic reductive pathway acting on both STT3A- and STT3B-dependent glycosylation. Our results provide further insight into the important role of the endoplasmic reticulum redox conditions in glycosylation site occupancy and demonstrate a link between redox conditions in the cytosol and glycosylation efficiency.