Cell entry of Simian Virus 40 (SV40) involves caveolar/lipid raft-mediated endocytosis, vesicular transport to the endoplasmic reticulum (ER), translocation into the cytosol, and import into the nucleus. We analyzed the effects of ER-associated processes and factors on infection and on isolated viruses and found that SV40 makes use of the thiol-disulfide oxidoreductases, ERp57 and PDI, as well as the retrotranslocation proteins Derlin-1 and Sel1L. ERp57 isomerizes specific interchain disulfides connecting the major capsid protein, VP1, to a crosslinked network of neighbors, thus uncoupling about 12 of 72 VP1 pentamers. Cryo-electron tomography indicated that loss of interchain disulfides coupled with calcium depletion induces selective dissociation of the 12 vertex pentamers, a step likely to mimic uncoating of the virus in the cytosol. Thus, the virus utilizes the protein folding machinery for initial uncoating before exploiting the ER-associated degradation machinery presumably to escape from the ER lumen into the cytosol.

The endoplasmic reticulum (ER) contains a number of thiol-disulfide oxidoreductases of the protein-disulfide isomerase (PDI) family that catalyze the formation of disulfide bonds in newly synthesized proteins. Here we describe the identification and characterization of a novel member of the human PDI family, TMX3 (thioredoxin-related transmembrane protein 3). The TMX3 gene encodes a protein of 454 amino acid residues that contains a predicted N-terminal signal sequence, a single domain with sequence similarity to thioredoxin and a CGHC active site sequence, a potential transmembrane domain, and a C-terminal KKKD tetrapeptide sequence that matches the classical KKXX-type consensus sequence for ER retrieval of type I transmembrane proteins. Endogenous TMX3 contains endoglycosidase Hsensitive glycans, localizes to the ER by immunofluorescence microscopy, and is present in the membrane fraction after alkaline extraction of the ER luminal content. The TMX3 transcript is found in a variety of tissues and is not up-regulated by the unfolded protein response. Circular dichroism spectroscopy of the recombinantly expressed luminal domain of TMX3 showed features typical of a properly folded protein of the ␣/␤ type. The redox potential of recombinant luminal TMX3 was determined to ؊0.157 V, similar to the values previously found for PDI and ERp57. Interestingly, TMX3 showed oxidase activity, and in human tissue-culture cells the protein was found partially in the oxidized form, potentially suggesting a function of the protein as a dithiol oxidase.The formation of disulfide bonds plays a critical role for the correct folding of most secretory and plasma membrane proteins in the endoplasmic reticulum (ER).1 Whereas folding of certain proteins proceeds by the sequential formation of native disulfides, it is clear that other proteins (e.g. the low density lipoprotein receptor) (1) form long range nonnative disulfides that are later rearranged as an integral folding step in living cells. Both the formation and the rearrangement of disulfide bonds often rely on the catalysis by thiol-disulfide oxidoreductases.Protein-disulfide isomerase (PDI) is the founding member of a family of thiol-disulfide oxidoreductases in the ER (reviewed in Ref.2). The protein contains four domains, named a, b, b, and a, all with homology to thioredoxin. Like this cytosolic reductase, the catalytic a and a domains of PDI contain tetrapeptide CXXC active site sequences (where X denotes any amino acid), for catalysis of thiol-disulfide exchange reactions. These reactions proceed through transient mixed disulfide intermediates between enzyme and substrate and lead to the oxidation (formation), reduction (breaking), or isomerization (rearrangement) of substrate cysteines. The two active site cysteines in oxidoreductases are found either in the oxidized disulfide or the reduced dithiol form; the disulfide form determines the function of the enzyme as an oxidase, whereas the dithiol form allows it to act as a reductase and/or an isomerase. Of the noncatalytic...

Disulfide bond formation in the endoplasmic reticulum is catalyzed by enzymes of the protein disulfide-isomerase family that harbor one or more thioredoxin-like domains. We recently discovered the transmembrane protein TMX3, a thiol-disulfide oxidoreductase of the protein disulfide-isomerase family. Here, we show that the endoplasmic reticulum-luminal region of TMX3 contains three thioredoxin-like domains, an N-terminal redox-active domain (named a) followed by two enzymatically inactive domains (b and b). Using the recombinantly expressed TMX3 domain constructs a, ab, and abb, we compared structural stability and enzymatic properties. By structural and biophysical methods, we demonstrate that the reduced a domain has features typical of a globular folded domain that is, however, greatly destabilized upon oxidization. Importantly, interdomain stabilization by the b domain renders the a domain more resistant toward chemical denaturation and proteolysis in both the oxidized and reduced form. In combination with molecular modeling studies of TMX3 abb, the experimental results provide a new understanding of the relationship between the multidomain structure of TMX3 and its function as a redox enzyme. Overall, the data indicate that in addition to their role as substrate and co-factor binding domains, redox-inactive thioredoxin-like domains also function in stabilizing neighboring redox-active domains.For many proteins of the secretory pathway and those targeted to the cell surface or secreted, correct folding depends on the formation of native disulfides. The formation of short range disulfide bonds found in some proteins likely requires little catalysis, whereas other proteins rely on the catalyzed formation and rearrangement of disulfides that must be introduced in a nontrivial pattern. The first ER enzyme found to promote these processes was PDI 3 (1). In vitro, PDI can catalyze oxidation of free cysteines as well as reduction and isomerization of incorrect disulfide bonds (2-4). It is now clear that a whole family of PDI-like proteins of almost 20 members exists in the mammalian ER (5, 6).The proteins of the PDI family all contain at least one domain with a thioredoxin fold, where the secondary structure elements are typically arranged as ␤ 1 -␣ 1 -␤ 2 -␣ 2 -␤ 3 -␣ 3 -␤ 4 -␤ 5 -␣ 4 . Thioredoxin-like domains that encompass a reactive CXXC tetrapeptide sequence are classified as a-type domains and otherwise as b-type domains. For instance, PDI contains four thioredoxin-like domains named a, b, b, and a, where a and a are redox-active, and b and b show no such activity. The redoxactive domains generally show a high level of sequence conservation, whereas the b-type domains display comparatively little sequence similarity. The function of many b-type domains remains unknown. It is clear, however, that the b domain in PDI confers substrate binding (7) and that the corresponding domain in ERp57, the closest homolog of PDI, interacts with the lectin chaperones calnexin and calreticulin that bind glycoprotein substrate...

The biosynthesis of small molecules can be fine-tuned by (re)engineering metabolic flux within cells. We have adapted this approach to optimize an in vivo selection system for the conversion of prephenate to phenylpyruvate, a key step in the production of the essential aromatic amino acid phenylalanine. Careful control of prephenate concentration in a bacterial host lacking prephenate dehydratase, achieved through provision of a regulable enzyme that diverts it down a parallel biosynthetic pathway, provides the means to systematically increase selection pressure on replacements of the missing catalyst. Successful differentiation of dehydratases whose activities vary over a >50,000-fold range and the isolation of mechanistically informative prephenate dehydratase variants from large protein libraries illustrate the potential of the engineered selection strain for characterizing and evolving enzymes. Our approach complements other common methods for adjusting selection pressure and should be generally applicable to any selection system that is based on the conversion of an endogenous metabolite. directed evolution ͉ enzyme design ͉ genetic complementation ͉ prephenate dehydratase ͉ tetracycline promoter

No abstract