Insoluble, inactive inclusion bodies are frequently formed upon recombinant protein production in transformed microorganisms. These inclusion bodies, which contain the recombinant protein in an highly enriched form, can be isolated by solid/liquid separation. After solubilization, native proteins can be generated from the inactive material by using in vitro folding techniques. New folding procedures have been developed for efficient in vitro reconstitution of complex hydrophobic, multidomain, oligomeric, or highly disulfide-bonded proteins. These protocols take into account process parameters such as protein concentration, catalysis of disulfide bond formation, temperature, pH, and ionic strength, as well as specific solvent ingredients that reduce unproductive side reactions. Modification of the protein sequence has been exploited to improve in vitro folding.

At normal temperatures, Hsp90 is one of the most abundant proteins in the cytosol of various eucaryotic cells. Upon heat shock, the level of Hsp90 is increased even more, suggesting that it is important for helping cells to survive under these conditions. However, studies so far have been almost exclusively concerned with the function of Hsp90 under non-stress conditions, and therefore only little is known about the role of Hsp90 during heat shock. As a model for heat shock in vitro, we have monitored the inactivation and subsequent aggregation of dimeric citrate synthase (CS) at elevated temperatures. Hsp90 effectively "stabilized" CS under conditions where the enzyme is normally inactivated and finally aggregates very rapidly. A kinetic dissection of the unfolding pathway of CS succeeded in revealing two intermediates which form and subsequently undergo irreversible aggregation reactions. Hsp90 apparently interacts transiently with these highly structured early unfolding intermediates. Binding and subsequent release of the intermediates favorably influences the kinetic partitioning between two competing processes, the further unfolding of CS and the productive refolding to the native state. As a consequence, CS is effectively stabilized in the presence of Hsp90. The significance of this interaction is especially evident in the suppression of aggregation, the major end result of thermal unfolding events in vivo and in vitro. These effects, which are ATP-independent, seem to be a general function of members of the Hsp90 family, since yeast and bovine Hsp90 as well as the Hsp90 homologue from Escherichia coli gave similar results. It seems likely that this function also reflects the role of Hsp90 under heat shock conditions in vivo. We therefore propose that members of the Hsp90 family convey thermotolerance by transiently binding to highly structured early unfolding intermediates, thereby preventing their irreversible aggregation and stabilizing the active species.

The lateral mobility of individual murine polyoma virus-like particles (VLPs) bound to live cells and artificial lipid bilayers was studied by single fluorescent particle tracking using total internal reflection fluorescence microscopy. The particle trajectories were analyzed in terms of diffusion rates and modes of motion as described by the moment scaling spectrum. Although VLPs bound to their ganglioside receptor in lipid bilayers exhibited only free diffusion, analysis of trajectories on live 3T6 mouse fibroblasts revealed three distinct modes of mobility: rapid random motion, confined movement in small zones (30 -60 nm in diameter), and confined movement in zones with a slow drift. After binding to the cell surface, particles typically underwent free diffusion for 5-10 s, and then they were confined in an actin filament-dependent manner without involvement of clathrin-coated pits or caveolae. Depletion of cholesterol dramatically reduced mobility of VLPs independently of actin, whereas inhibition of tyrosine kinases had no effect on confinement. The results suggested that clustering of ganglioside molecules by the multivalent VLPs induced transmembrane coupling that led to confinement of the virus͞receptor complex by cortical actin filaments.gangliosides ͉ lipid raft ͉ total internal reflection fluorescence microscopy ͉ virus entry ͉ confinement zone

The yeast cytosol contains multiple homologs of the DnaK and DnaJ chaperone family. Our current understanding of which homologs functionally interact is incomplete. Zuotin is a DnaJ homolog bound to the yeast ribosome. We have now identified the DnaK homolog Ssz1p͞Pdr13p as zuotin's partner chaperone. Zuotin and Ssz1p form a ribosome-associated complex (RAC) that is bound to the ribosome via the zuotin subunit. RAC is unique among the eukaryotic DnaK-DnaJ systems, as the 1:1 complex is stable, even in the presence of ATP or ADP. In vitro, RAC stimulates the translocation of a ribosome-bound mitochondrial precursor protein into mitochondria, providing evidence for its chaperonelike effect on nascent chains. In agreement with the existence of a functional complex, deletion of each RAC subunit resulted in a similar phenotype in vivo. However, overexpression of zuotin partly rescued the growth defect of the ⌬ssz1 strain, whereas overexpression of Ssz1p did not affect the ⌬zuo1 strain, suggesting a pivotal function for the DnaJ homolog. P roteins synthesized on cytosolic ribosomes either fold or translocate to their final location cotranslationally or posttranslationally. Translocation and folding require chaperonelike proteins that often serve multiple and overlapping functions. The complex chaperone network in the eukaryotic cell is currently only poorly understood (1-3).Most posttranslational translocation events require the translocating proteins to be unfolded. This requirement is at least partly ensured by binding of cytosolic chaperones to newly translated proteins. The yeast DnaK͞Hsp70 homolog Ssa1͞2p and its partner protein, the DnaJ homolog Ydj1p, are involved in protein translocation into a variety of compartments (4). Besides their role in translocation, Ssa1͞2p and Ydj1p are involved in cytosolic protein folding, most likely in a posttranslational manner (5-7). Other chaperones assisting posttranslational folding in the eukaryotic cytosol are the chaperonin CCT and Hsp90 (2,8,9).Some chaperones interact cotranslationally with polypeptides. In both eukaryotes and prokaryotes, soluble DnaK and DnaJ homologs bind to nascent chains and subsequently assist their folding (2, 10, 11). The ribosome-bound DnaK homolog Ssb1͞2p can be crosslinked to nascent chains, providing evidence for a functionally important interaction (12).There is long-standing circumstantial evidence of cotranslational import into mitochondria (13). More recent data suggest that some precursor proteins even require a cotranslational mechanism to be imported into mitochondria (14-16). However, no specialized component of a mitochondrial cotranslational translocation system, comparable to a signal recognition particle or signal recognition particle receptor, has been identified (17).In a previous study we introduced an in vitro mitochondrial protein import assay for the identification of cytosolic components involved in either cotranslational translocation or interacting with nascent precursor proteins in a chaperone-like manner. The assay, b...