Recombinant production of membrane proteins in yeast.

Biochemical pathways are compartmentalized in living cells. This permits each cell to maintain chemical compositions that differ between the cytosol, intracellular organelles and the external environment. Achieving this requires each compartment to be very selective in what is allowed to enter and leave. Nature has solved this by surrounding each cell and each organelle with a virtually solute impermeable lipid membrane, embedded with integral membrane proteins that mediate strictly controlled trans-membrane movement of matter and information. Access to pure and active integral membrane proteins is therefore required to comprehend membrane biology, ultimately through high-resolution structures of the membrane proteome and, therefore, also for our understanding of physiology. Unfortunately, apart from a few exceptions, membrane proteins cannot be purified from native tissue but need to be produced recombinantly, which is eminently challenging. This chapter shows how we have engineered yeast to provide high levels of prime quality membrane proteins of prokaryotic, archaeal or eukaryotic origin for structural biology.

Saccharomyces cerevisiae as a superior host for overproduction of prokaryotic integral membrane proteins.

Integral membrane proteins (IMPs) constitute ~30% of all proteins encoded by the genome of any organism and Escherichia coli remains the first-choice host for recombinant production of prokaryotic IMPs. However, the expression levels of prokaryotic IMPs delivered by this bacterium are often low and overproduced targets often accumulate in inclusion bodies. The targets are therefore often discarded to avoid an additional and inconvenient refolding step in the purification protocol. Here we compared expression of five prokaryotic (bacterial and archaeal) IMP families in E. coli and Saccharomyces cerevisiae. We demonstrate that our S. cerevisiae-based production platform is superior in expression of four investigated IMPs, overall being able to deliver high quantities of active target proteins. Surprisingly, in case of the family of zinc transporters (Zrt/Irt-like proteins, ZIPs), S. cerevisiae rescued protein expression that was undetectable in E. coli. We also demonstrate the effect of localization of the fusion tag on expression yield and sample quality in detergent micelles. Lastly, we present a road map to achieve the most efficient expression of prokaryotic IMPs in our yeast platform. Our findings demonstrate the great potential of S. cerevisiae as host for high-throughput recombinant overproduction of bacterial and archaeal IMPs for downstream biophysical characterization.

Author Correction: Characterization of Hailey-Hailey Disease-mutants in presence and absence of wild type SPCA1 using Saccharomyces cerevisiae as model organism.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Characterization of Hailey-Hailey Disease-mutants in presence and absence of wild type SPCA1 using Saccharomyces cerevisiae as model organism.

Hailey-Hailey disease is an autosomal genetic disease caused by mutations in one of the two ATP2C1 alleles encoding the secretory pathway Ca2+/Mn2+-ATPase, hSPCA1. The disease almost exclusively affects epidermis, where it mainly results in acantholysis of the suprabasal layers. The etiology of the disease is complex and not well understood. We applied a yeast based complementation system to characterize fourteen disease-causing ATP2C1 missense mutations in presence or absence of wild type ATP2C1 or ATP2A2, encoding SERCA2. In our yeast model system, mutations in ATP2C1 affected Mn2+ transport more than Ca2+ transport as twelve out of fourteen mutations were unable to complement Mn2+ sensitivity while thirteen out of fourteen to some extent complemented the high Ca2+requirement. Nine out of fourteen mutations conferred a cold sensitive complementation capacity. In absence of a wild type ATP2C1 allele, twelve out of fourteen mutations induced an unfolded protein response indicating that in vivo folding of hSPCA1 is sensitive to disease causing amino acid substitutions and four of the fourteen mutations caused the hSPCA1 protein to accumulate in the vacuolar membrane. Co-expression of either wild type ATP2C1 or ATP2A2 prevented induction of the unfolded protein response and hSPCA1 mis-localization.

Structure of the human ClC-1 chloride channel.

ClC-1 protein channels facilitate rapid passage of chloride ions across cellular membranes, thereby orchestrating skeletal muscle excitability. Malfunction of ClC-1 is associated with myotonia congenita, a disease impairing muscle relaxation. Here, we present the cryo-electron microscopy (cryo-EM) structure of human ClC-1, uncovering an architecture reminiscent of that of bovine ClC-K and CLC transporters. The chloride conducting pathway exhibits distinct features, including a central glutamate residue ("fast gate") known to confer voltage-dependence (a mechanistic feature not present in ClC-K), linked to a somewhat rearranged central tyrosine and a narrower aperture of the pore toward the extracellular vestibule. These characteristics agree with the lower chloride flux of ClC-1 compared with ClC-K and enable us to propose a model for chloride passage in voltage-dependent CLC channels. Comparison of structures derived from protein studied in different experimental conditions supports the notion that pH and adenine nucleotides regulate ClC-1 through interactions between the so-called cystathionine-ß-synthase (CBS) domains and the intracellular vestibule ("slow gating"). The structure also provides a framework for analysis of mutations causing myotonia congenita and reveals a striking correlation between mutated residues and the phenotypic effect on voltage gating, opening avenues for rational design of therapies against ClC-1-related diseases.