Mitochondrial energy-transducing nicotinamide nucleotide transhydrogenase. Purification and properties of the proteinase K-bisected enzyme.

The mitochondrial proton-translocating nicotinamide nucleotide transhydrogenase is embedded in the inner membrane as a homodimer of monomer Mr = 109,288. Its N-terminal 430 residues and C-terminal 200 residues protrude into the matrix, whereas its central 400 residues appear to intercalate into the inner membrane as 14 hydrophobic clusters of about 20 residues each (Yamaguchi, M., and Hatefi, Y. (1991) J. Biol. Chem. 266, 5728-5735). Treatment of mitoplasts (mitochondria denuded of outer membrane) with several proteolytic enzymes cleaves the transhydrogenase into a 72-kDa N-terminal and a 37-kDa C-terminal fragment. The cleavage site of proteinase K was determined to be Ala690-Ala691, which is located in a small loop of the transhydrogenase exposed on the cytosolic side of the inner membrane. This paper shows that the bisected transhydrogenase can be purified from proteinase K-treated mitoplasts with retention of greater than or equal to 85% transhydrogenase activity. The inactivation rate of the bisected enzyme by trypsin and N-ethylmaleimide was altered in the presence of NADP and NADPH, suggesting substrate-induced conformation changes similar to those reported previously for the intact transhydrogenase. Also, like the intact enzyme, proteoliposomes of the bisected transhydrogenase were capable of membrane potential formation and internal acidification coupled to NADPH----NAD transhydrogenation. The properties of the bisected transhydrogenase have been discussed in relation to those of the two-subunit Escherichia coli transhydrogenase, the bisected lac permease (via gene restriction), and the fragmented and reconstituted bacteriorhodopsin.     

The orientation of transhydrogenase in the inner mitochondrial membrane of rat liver

The orientation of the transmembranous enzyme, pyridine dinucleotide transhydrogenase, in the inner mitochondrial membrane of rat liver has been determined by evaluating effects of proteases on the integrity of the enzyme in mitoplasts and submitochondrial particles. Following treatment of these membranes with the nonspecific protease, proteinase K, antigenic proteolytic products were detected by immunoblot analysis using polyclonal antibody prepared against purified bovine heart enzyme. Proteinase K treatment of mitoplasts converted the 110,000 transhydrogenase monomer into a single immunoreactive species having Mr 75,000. This proteolytic product is stable to further incubation with the protease. Treatment of submitochondrial particles with proteinase K resulted in the disappearance of the 110,000 monomer and the transient formation of an intermediate product with Mr 52,000. Information from these proteolysis studies was used to construct a model of the orientation of transhydrogenase in the inner mitochondrial membrane. This model indicates that transhydrogenase (Mr 110,000) contains a core of proteolytically inaccessible proteins within the membrane (Mr 23,000) bounded by extramembranous domains on the matrix (Mr 52,000) and cytoplasmic (Mr 35,000) face of the inner mitochondrial membrane.     

Mitochondrial energy-linked nicotinamide nucleotide transhydrogenase: effect of substrates on the sensitivity of the enzyme to trypsin and identification of tryptic cleavage sites

The mitochondrial nicotinamide nucleotide transhydrogenase catalyzes hydride ion transfer between NAD(H) and NADP(H) in a reaction that is coupled to proton translocation across the inner mitochondrial membrane. The enzyme (1043 residues) is composed of an N-terminal hydrophilic segment (approximately 400 residues long) which binds NAD(H), a C-terminal hydrophilic segment (approximately 200 residues long) which binds NADP(H), and a central hydrophobic segment (approximately 400 residues long) which appears to form about 14 membrane-intercalating clusters of approximately 20 residues each. Substrate modulation of transhydrogenase conformation appears to be intimately associated with its mechanism of proton translocation. Using trypsin as a probe of enzyme conformation change, we have shown that NADPH (and to a much lesser extent NADP) binding alters transhydrogenase conformation, resulting in increased susceptibility of several bonds to tryptic hydrolysis. NADH and NAD had little or no effect, and the NADPH concentration for half-maximal enhancement of trypsin sensitivity of transhydrogenase activity (35 microM) was close to the Km of the enzyme for NADPH. The NADPH-promoted trypsin cleavage sites were located 200-400 residues distant from the NADP(H) binding domain near the C-terminus. For example, NADPH binding greatly increased the trypsin sensitivity of the K410-T411 bond, which is separated from the NADP(H) binding domain by the 400-residue-long membrane-intercalating segment. It also enhanced the tryptic cleavage of the R602-L603 bond, which is located within the central hydrophobic segment. These results, which suggest a protein conformation change as a result of NADPH binding, have been discussed in relation to the mechanism of proton translocation by the transhydrogenase.     

Topological analysis of the pyridine nucleotide transhydrogenase of Escherichia coli using proteolytic enzymes

The pyridine nucleotide transhydrogenase of Escherichia coli has an alpha 2 beta 2 structure (alpha: Mr, 54,000; beta: Mr, 48,700). Hydropathy analysis of the amino acid sequences suggested that the 10 kDa C-terminal portion of the alpha subunit and the N-terminal 20-25 kDa region of the beta subunit are composed of transmembranous alpha-helices. The topology of these subunits in the membrane was investigated using proteolytic enzymes. Trypsin digestion of everted cytoplasmic membrane vesicles released a 43 kDa polypeptide from the alpha subunit. The beta subunit was not susceptible to trypsin digestion. However, it was digested by proteinase K in everted vesicles. Both alpha and beta subunits were not attacked by trypsin and proteinase K in right-side out membrane vesicles. The beta subunit in the solubilized enzyme was only susceptible to digestion by trypsin if the substrates NADP(H) were present. NAD(H) did not affect digestion of the beta subunit. Digestion of the beta subunit of the membrane-bound enzyme by trypsin was not induced by NADP(H) unless the membranes had been previously stripped of extrinsic proteins by detergent. It is concluded that binding of NADP(H) induces a conformational change in the transhydrogenase. The location of the trypsin cleavage sites in the sequences of the alpha and beta subunits were determined by N- and C-terminal sequencing. A model is proposed in which the N-terminal 43 kDa region of the alpha subunit and the C-terminal 30 kDa region of the beta subunit are exposed on the cytoplasmic side of the inner membrane of E. coli. Binding sites for pyridine nucleotide coenzymes in these regions were suggested by affinity chromatography on NAD-agarose columns.     

Targeting and membrane-insertion of a sunflower oleosin in vitro and in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>: the central hydrophobic domain contains more than one signal sequence,and directs oleosin insertion into the endoplasmic reticulum membrane using a signal anchor sequence mechanism

A range of N- and C-terminal deletions of an oleosin from Helianthus annuus L. were used to study the endoplasmic reticulum (ER) targeting and membrane insertion of this protein both in vitro and in vivo in yeast ( Saccharomyces cerevisiae). Neither the N- nor the C-terminal hydrophilic domains are important for targeting and/or membrane insertion, with all the information required for these processes located within the central hydrophobic region of the protein. However, in vitro membrane-insertion experiments suggest that these domains are important for a correct topology of the oleosin within the ER membrane. The first half of the hydrophobic central domain, flanked by the positively charged N-terminal domain, is likely to function as a type-II signal-anchor (SAII) sequence. However, in the absence of the N-terminal 26 residues of this domain, the proline-knot region and the second half of this hydrophobic domain are sufficient to direct oleosin to the ER and to allow stable (but far less efficient) integration of the protein into the membrane. Taken together, these results indicate that oleosin contains more than one domain that is capable of interacting with the signal recognition particle to direct the protein to the ER membrane.     

The mitochondrial tricarboxylate carrier of silver eel: dimeric structure and cytosolic exposure of both N- and C-termini

The mitochondrial tricarboxylate carrier plays a fundamental role in the hepatic fatty acid synthesis. In this study, we investigated the transmembrane organization of this protein in the inner membrane of eel liver mitochondria using anti–N-terminal and anti–C-terminal antibodies. These antibodies recognized the N- and C-termini of the tricarboxylate carrier in intact mitoplasts, thus suggesting a cytosolic exposure of these regions in the membrane-bound protein. This structural arrangement of the tricarboxylate carrier was further confirmed by protease treatment of intact mitoplasts. Moreover, the oligomeric state of the native tricarboxylate carrier was investigated by blue native electrophoresis. A dimeric form of the carrier protein was found when eel liver mitochondria were solubilized with the mild detergent digitonin. These findings suggest an arrangement of the dimeric tricarboxylate carrier into an even number of membrane-spanning domains, with the N-terminal and C-terminal regions oriented toward the intermembrane space of fish mitochondria.     

Membrane topology of gamma-secretase component PEN-2

PEN-2 is an integral membrane protein that is a necessary component of the gamma-secretase complex, which is central in the pathogenesis of Alzheimer's disease and is also required for Notch signaling. In the absence of PEN-2, Notch signaling fails to guide normal development in Caenorhabditis elegans, and amyloid beta peptide is not generated from the amyloid precursor protein. Human PEN-2 is a 101-amino acid protein containing two putative transmembrane domains. To understand its interaction with other gamma-secretase components, it is important to know the membrane topology of each member of the complex. To characterize the membrane topology of PEN-2, we introduced single amino acid changes in each of the three hydrophilic regions of PEN-2 to generate N-linked glycosylation sites. We found that the N-linked glycosylation sites present in the N- and C-terminal domains of PEN-2 were utilized, whereas a site in the hydrophilic "loop" region connecting the two transmembrane domains was not. The addition of a carbohydrate structure in the N-terminal domain of PEN-2 prevented association with presenilin 1, whereas glycosylation in the C-terminal region of PEN-2 did not, suggesting that the N-terminal domain is important for interactions with presenilin 1. Immunofluorescence microscopy with selective permeabilization of the plasma membrane of cells expressing epitope-tagged forms of PEN-2 confirmed the lumenal location of both the N and C termini. A protease protection assay also demonstrated that the loop domain of PEN-2 is cytosolic. Thus, PEN-2 spans the membrane twice, with the N and C termini facing the lumen of the endoplasmic reticulum.     

N-218 MLN64, a protein with StAR-like steroidogenic activity, is folded and cleaved similarly to StAR

The steroidogenic acute regulatory protein (StAR) facilitates the movement of cholesterol from the outer to inner mitochondrial membrane in adrenal and gonadal cells, fostering steroid biosynthesis. MLN64 is a 445-amino acid protein of unknown function. When 218 amino-terminal residues of MLN-64 are deleted, the resulting N-218 MLN64 has 37% amino acid identity with StAR and 50% of StAR's steroidogenic activity in transfected cells. Antiserum to StAR cross-reacts with N-218 MLN64, indicating the presence of similar epitopes in both proteins. Western blotting shows that MLN64 is proteolytically cleaved in the placenta to a size indistinguishable from N-218 MLN64. Bacterially expressed N-218 MLN64 exerts StAR-like activity to promote the transfer of cholesterol from the outer to inner mitochondrial membrane in vitro. CD spectroscopy indicates that N-218 MLN64 is largely alpha-helical and minimally affected by changes in ionic strength or the hydrophobic character of the solvent, although glycerol increases the beta-sheet content. However, decreasing pH diminishes structure, causing aggregation. Limited proteolysis at pH 8.0 shows that the C-terminal domain of N-218 MLN64 is accessible to proteolysis whereas the 244-414 domain is resistant, suggesting it is more compactly folded. The presence of a protease-resistant domain and a protease-sensitive carboxy-terminal domain in N-218 MLN64 is similar to the organization of StAR. However, as MLN64 never enters the mitochondria, the protease-resistant domain of MLN64 cannot be a mitochondrial pause-transfer sequence, as has been proposed for StAR. Thus the protease-resistant domain of N-218 MLN64, and by inference the corresponding domain of StAR, may have direct roles in their action to foster the flux of cholesterol from the outer to the inner mitochondrial membrane.     

A eukaryotic carboxyl-terminal signal sequence translocating large hydrophilic domains across membranes

Yeast Golgi ecto-ATPase Ynd1p is an unusual type III membrane protein with the longest translocated N-terminus reported. Sequential deletion analysis reveals that translocation of this 500-residue-long hydrophilic domain across the membranes requires the C-terminal transmembrane domain of Ynd1p and its flanking regions. Additional studies indicate that the topogenic sequence of Ynd1p overrides the effect of a reverse signal-anchor sequence present at the N-terminus of Ynd1p, while it is not affected by a classic signal sequence at the N-terminus. When placed at the C-terminal end, the sequence can translocate large extracellular domains of two membrane proteins across the membranes. The data demonstrate the existence of a true eukaryotic C-terminal signal sequence.     

A class of amphipathic proteins associated with lipid storage bodies in plants. Possible similarities with animal serum apolipoproteins

The lipid-storing tissues of plants contain many small (0.2-1 microns) lipid (normally triacylglycerol) droplets which are surrounded and stabilized by a mixed phospholipid and protein annulus. The proteinaceous components of the lipid storage bodies are termed oleosins and are not associated with any other cellular structures. The major oleosins of rapeseed and radish have been isolated by preparative SDS-PAGE and are respectively classes of 19 kDa and 20 kDa proteins. Both protein classes were N-terminally blocked for direct sequencing, but were partially sequenced following limited proteolytic digestion. The major rapeseed oleosin was made up of at least two 19 kDa polypeptides, termed nap-I and nap-II, which have closely related but different amino acid sequences. A single 20 kDa oleosin, termed rad-I, was found in radish. A near full length cDNA clone for a major rapeseed oleosin was sequenced and found to correspond almost exactly to the sequence of nap-II. The sequences of nap-I and rad-I show very close similarity to one another, as do the sequences of nap-II and the previously determined sequence for the major oleosin from maize. All four oleosins have a large central hydrophobic domain flanked by polar N- and C-terminal domains. Secondary structure predictions for the four oleosins are similar and a novel model is proposed based on a central hydrophobic beta-strand region flanked by an N-terminal polar alpha-helix and a C-terminal amphipathic alpha-helix. The possibility that oleosins exhibit structural and functional similarities with some animal apolipoproteins is discussed.     

Pediocin-like antimicrobial peptides (class IIa bacteriocins) and their immunity proteins: biosynthesis, structure, and mode of action.

Pediocin-like antimicrobial peptides (AMPs) form a group of lactic acid bacteria produced, cationic membrane-permeabilizing peptides with 37 to 48 residues. Upon exposure to membrane-mimicking entities, their hydrophilic, cationic, and highly conserved N-terminal region forms a three-stranded antiparallel beta-sheet supported by a conserved disulfide bridge. This N-terminal beta-sheet region is followed by a central amphiphilic alpha-helix and this in most (if not all) of these peptides is followed by a rather extended C-terminal tail that folds back onto the central alpha-helix, thereby creating a hairpin-like structure in the C-terminal half. There is a flexible hinge between the beta-sheet N-terminal region and the hairpin C-terminal region and one thus obtains two domains that may move relative to each other. The cationic N-terminal beta-sheet domain mediates binding of the pediocin-like AMPs to the target-cell surface through electrostatic interactions, while the more hydrophobic and amphiphilic C-terminal hairpin domain penetrates into the hydrophobic part of the target-cell membrane, thereby mediating leakage through the membrane. The hinge provides the structural flexibility that enables the C-terminal hairpin domain to dip into the hydrophobic part of the membrane. Despite extensive sequence similarities, these AMPs differ markedly in their target-cell specificity, and results obtained with hybrid AMPs indicate that the membrane-penetrating hairpin-like C-terminal domain is the major specificity determinant.Bacteria that produce pediocin-like AMPs also produce a 11-kDa cognate immunity protein that protects the producer. The immunity proteins are well-structured, 4-helix bundle cytosolic proteins. They show a high degree of specificity in that they largely recognize and confer immunity only to their cognate AMP and in some cases to a few AMPs that are closely related to their cognate AMP. The C-terminal half of the immunity proteins contains a domain that is involved in specific recognition of the C-terminal membrane-penetrating specificity-determining hairpin domain of the cognate AMP.     

Structure, function and tissue forms of the C-terminal globular domain of collagen XVIII containing the angiogenesis inhibitor endostatin.

The C-terminal domain NC1 of mouse collagen XVIII (38 kDa) and the shorter mouse and human endostatins (22 kDa) were prepared in recombinant form from transfected mammalian cells. The NC1 domain aggregated non-covalently into a globular trimer which was partially cleaved by endogenous proteolysis into several monomers (25-32 kDa) related to endostatin. Endostatins were obtained in a highly soluble, monomeric form and showed a single N-terminal sequence which, together with other data, indicated a compact folding. Endostatins and NC1 showed a comparable binding activity for the microfibrillar fibulin-1 and fibulin-2, and for heparin. Domain NC1, however, was a distinctly stronger ligand than endostatin for sulfatides and the basement membrane proteins laminin-1 and perlecan. Immunological assays demonstrated endostatin epitopes on several tissue components (22-38 kDa) and in serum (120-300 ng/ml), the latter representing the smaller variants. The data indicated that the NC1 domain consists of an N-terminal association region (approximately 50 residues), a central protease-sensitive hinge region (approximately 70 residues) and a C-terminal stable endostatin domain (approximately 180 residues). They also demonstrated that proteolytic release of endostatin can occur through several pathways, which may lead to a switch from a matrix-associated to a more soluble endocrine form.     

Molecular organization (topography) of cytochrome P-450(11)beta in mitochondrial membrane and phospholipid vesicles as studied by trypsinolysis

Cytochrome P-450(11)beta from adrenal cortex is an intrinsic membrane protein embedded in the inner mitochondrial membrane. Topography of the protein inside a phospholipid bilayer was examined using controlled proteolysis of purified cytochrome P-450(11)beta following its integration into artificial liposomes. Inclusion of the protein into phospholipid vesicles led to a marked stabilization of the cytochrome activity. Trypsin treatment of the liposome-integrated cytochrome resulted in the rapid disappearance of the native protein moiety (47 kDa), while a major 34 kDa peptide component was formed. This peptide core retained the heme moiety and part of the cytochrome steroid-11 beta hydroxylase activity. Very similar observations were obtained when inside-out vesicles prepared from isolated adrenocortical mitoplasts were examined with the same approach. It is thus suggested that adrenocortical cytochrome P-450(11)beta is embedded in the inner mitochondrial membrane as well as in artificial liposomes by a major hydrophobic domain associated with the heme moiety while a limited domain remains accessible on the matrix side of the membrane surface. The previous described phosphorylation of the cytochrome P-450(11)beta on a serine residue, by the cAMP-dependent protein kinase is suggested to occur in the protein domain oriented toward the membrane surface, the phosphorylation site being lost under mild proteolytic digestion of the membrane-integrated protein.     

The primary structure of the mitochondrial energy-linked nicotinamide nucleotide transhydrogenase deduced from the sequence of cDNA clones

The amino acid sequence of the bovine mitochondrial nicotinamide nucleotide transhydrogenase, which catalyzes hydride ion transfer between NAD(H) and NADP(H) coupled to proton translocation across the mitochondrial inner membrane, has been deduced from the corresponding cDNA. Two clones were isolated by screening a bovine lambda gt10 cDNA library, using two synthetic oligonucleotides and a cDNA restriction fragment as probes. The inserts together covered 3,105 base pairs of coding sequence, corresponding to 1.035 amino acid residues. However, the reading frame at the 5' end was still open. N-terminal sequence analysis of the isolated enzyme indicated the presence of 8 additional residues. Thus, the mature transhydrogenase appeared to have 1,043 amino acid residues and a calculated molecular weight of 109,212. The deduced amino acid sequence of the transhydrogenase contained the sequences of four tryptic peptides that had been isolated from the enzyme. Two of these were the peptides that had been used for construction of the oligonucleotide probes. The other two were tryptic peptides isolated after labeling the NAD-binding site of the transhydrogenase once with [3H]p-fluorosulfonylbenzoyl-5'-adenosine (FSBA), and another time with [14C]N,N'-dicyclohexylcarbodiimide. The FSBA-labeled peptide was found to be located immediately upstream of the [14C]N,N'-dicyclohexylcarbodiimide-labeled peptide, about 230 residues from the N terminus. One of the tryptic peptides used for oligonucleotide probe construction was the same as that labeled with [3H]FSBA when the NAD-binding site was protected from FSBA attack. This peptide, which might be at the NADP-binding site of the transhydrogenase, was located very near the C terminus of the enzyme. The central region of the transhydrogenase (residues 420-850) is highly hydrophobic and appears to comprise about 14 membrane-spanning segments. By comparison, the N- and the C-terminal regions of the enzyme, which contain the NAD- and the putative NADP-binding sites, respectively, are relatively hydrophilic and are probably located outside the mitochondrial inner membrane on the matrix side. There is considerable homology between the bovine enzyme and the Escherichia coli transhydrogenase (two subunits, alpha with Mr = 54,000 and beta with Mr = 48,700), whose amino acid sequence has been determined from the genes (Clarke, D.M., Loo, T.W., Gillam, S., and Bragg, P.D. (1986) Eur. J. Biochem. 158, 647-653).     

Membrane protein topology of oleosin is constrained by its long hydrophobic domain.

Oleosin proteins from Arabidopsis assume a unique endoplasmic reticulum (ER) topology with a membrane-integrated hydrophobic (H) domain of 72 residues, flanked by two cytosolic hydrophilic domains. We have investigated the targeting and topological determinants present within the oleosin polypeptide sequence using ER-derived canine pancreatic microsomes. Our data indicate that oleosins are integrated into membranes by a cotranslational, translocon-mediated pathway. This is supported by the identification of two independent functional signal sequences in the H domain, and by demonstrating the involvement of the SRP receptor in membrane targeting. Oleosin topology was manipulated by the addition of an N-terminal cleavable signal sequence, resulting in translocation of the N terminus to the microsomal lumen. Surprisingly, the C terminus failed to translocate. Inhibition of C-terminal translocation was not dependent on either the sequence of hydrophobic segments in the H domain, the central proline knot motif or charges flanking the H domain. Therefore, the topological constraint results from the length and/or the hydrophobicity of the H domain, implying a general case that long hydrophobic spans are unable to translocate their C terminus to the ER lumen.     

Deletion of Various Carboxy-Terminal Domains of Lactococcus lactis SK11 Proteinase: Effects on Activity, Specificity, and Stability of the Truncated Enzyme

The Lactococcus lactis SK11 cell envelope proteinase is an extracellular, multidomain protein of nearly 2,000 residues consisting of an N-terminal serine protease domain, followed by various other domains of largely unknown function. Using a strategy of deletion mutagenesis, we have analyzed the function of several C-terminal domains of the SK11 proteinase which are absent in cell envelope proteinases of other lactic acid bacteria. The various deletion mutants were functionally expressed in L. lactis and analyzed for enzyme stability, activity, (auto)processing, and specificity toward several substrates. C-terminal deletions of first the cell envelope W (wall) and AN (anchor) domains and then the H (helix) domain leads to fully active, secreted proteinases of unaltered specificity. Gradually increasing the C-terminal deletion into the so-called B domain leads to increasing instability and autoproteolysis and progressively less proteolytic activity. However, the mutant with the largest deletion (838 residues) from the C terminus and lacking the entire B domain still retains proteolytic activity. All truncated enzymes show unaltered proteolytic specificity toward various substrates. This suggests that the main role played by these domains is providing stability or protection from autoproteolysis (B domain), spacing away from the cell (H domain), and anchoring to the cell envelope (W and AN domains). In addition, this study allowed us to more precisely map the main C-terminal autoprocessing site of the SK11 proteinase and the epitope for binding of group IV monoclonal antibodies.     

In the uncoupling protein from brown adipose tissue the C-terminus protrudes to the c-side of the membrane as shown by tryptic cleavage

Limited proteolytic digestion of the uncoupling protein (UCP) with trypsin yielded a cleavage product only about 2 kDa smaller than the original UCP (33 kDa). This cleavage can be obtained with the solubilized isolated protein detergent micelle as well as in original brown adipose mitochondria. The cleavage site is identified by C-terminal sequence to be located near the C-terminus at lysine 292. This C-terminus, a 10 residue long peptide, is strongly hydrophilic and can be expected to be localized outside the membrane. In UCP this C-terminal stretch represents a structural difference to the similarly folded ADP/ATP carrier which does not form a corresponding cleavage product. Comparison of tryptic cleavage of UCP in mitochondria with differently broken outer membrane, in sonic particles of mitochondria, as well as in UCP proteoliposomes, indicate that the C-terminus is directed versus the cytosolic site of the membrane. Because of the easy susceptibility to trypsin, the cleavage site must be surface-exposed and the C-terminal section unusually mobile.     

Mutagenesis of the yellow fever virus NS2B protein: effects on proteolytic processing, NS2B-NS3 complex formation, and viral replication.

To study the role of specific regions of the yellow fever virus NS2B protein in proteolytic processing and association with the NS3 proteinase domain, a series of mutations were created in the hydrophobic regions and in a central conserved hydrophilic region proposed as a domain important for NS2B function. The effects of these mutations on cis cleavage at the 2B/3 cleavage site and on processing at other consensus cleavage sites for the NS3 proteinase in the nonstructural region were then characterized by cell-free translation and transient expression in BHK cells. Association between NS2B and the NS3 proteinase domain and the effects of mutations on complex formation were investigated by nondenaturing immunoprecipitation of these proteins expressed in infected cells, by cell-free translation, or by recombinant vaccinia viruses. Mutations within the hydrophobic regions had subtle effects on proteolytic processing, whereas mutations within the conserved domain dramatically reduced cleavage efficiency or abolished all cleavages. The conserved domain of NS2B is also implicated in formation of an NS2B-NS3 complex on the basis of the ability of mutations in this region to eliminate both association of these two proteins and trans-cleavage activity. In addition, mutations which either eliminated proteolytic processing or had no apparent effect on processing were found to abolish recovery of infectious virus following RNA transfection. These results suggest that the conserved region of NS2B is a domain essential for the function of the NS3 proteinase. Hydrophobic regions of NS2B whose structural integrity may not be essential for proteolytic processing may have additional functions during viral replication.