Effects of CapZ, an actin capping protein of muscle, on the polymerization of actin

We have studied the interaction of CapZ, a barbed-end actin capping protein from the Z line of skeletal muscle, with actin. CapZ blocks actin polymerization and depolymerization (i.e., it "caps") at the barbed end with a Kd of approximately 0.5-1 nM or less, measured by three different assays. CapZ inhibits the polymerization of ATP-actin onto filament ends with ATP subunits slightly less than onto ends with ADP subunits, and onto ends with ADP-BeF3- subunits about as much as ends with ADP subunits. No effect of CapZ is seen at the pointed end by measurements either of polymerization from acrosomal processes or of the critical concentration for polymerization at steady state. CapZ has no measureable ability to sever actin filaments in a filament dilution assay. CapZ nucleates actin polymerization at a rate proportional to the first power of the CapZ concentration and the 2.5 power of the actin concentration. No significant binding is observed between CapZ and rhodamine-labeled actin monomers by fluorescence photobleaching recovery. These new experiments are consistent with but do not distinguish between three models for nucleation proposed previously (Cooper & Pollard, 1985). As a prelude to the functional studies, the purification protocol for CapZ was refined to yield 2 mg/kg of chicken breast muscle in 1 week. The activity is stable in solution and can be lyophilized. The native molecular weight is 59,600 +/- 2000 by equilibrium ultracentrifugation, and the extinction coefficient is 1.25 mL mg-1 cm-1 by interference optics. Polymorphism of the alpha and beta subunits has been detected by isoelectric focusing and reverse-phase chromatography. CapZ contains no phosphate (less than 0.1 mol/mol).     

Characterization of alpha 2-macroglobulin-plasmin complexes: complete subunit cleavage alters receptor recognition in vivo and in vitro

When human alpha 2-macroglobulin (alpha 2M) binds proteinases, it undergoes subunit cleavage. Binding of small proteinases such as trypsin results in proteolysis of each of the four subunits of the inhibitor. By contrast, previous studies suggest that reaction of plasmin with alpha 2M results in cleavage of only two or three of the inhibitor subunits. In this paper, we demonstrate that the extent of subunit cleavage of alpha 2M is a function of plasmin concentration. When alpha 2M was incubated with a 2.5-fold excess of plasmin, half of the subunits were cleaved; however, at a 20-fold enzyme to inhibitor ratio, greater than 90% of the subunits were cleaved with no additional plasmin binding. This increased cleavage was catalyzed by free rather than bound plasmin. It is concluded that this "nonproductive" subunit cleavage is dependent upon the molar ratio of proteinase to inhibitor. The consequence of complete subunit cleavage on receptor recognition of alpha 2M-plasmin (alpha 2M-Pm) complexes was studied. Preparations of alpha 2M-Pm with only two cleaved subunits bound to the murine macrophage receptor with a Kd of 0.4 nM and 60 fmol of bound complex/mg of cell protein. When preparations of alpha 2-M-Pm with four cleaved subunits were studied, the Kd was unaltered but ligand binding increased to 140 fmol/mg of cell protein. The receptor binding behavior of the latter preparation is equivalent to that observed when alpha 2M is treated with small proteinases such as trypsin. This study suggests that receptor recognition site exposure is not complete in the alpha 2M-Pm complex with half of the subunits cleaved. Proteolytic cleavage of the remaining subunits of the inhibitor results in a further conformational change exposing the remaining receptor recognition sites.     

Polymerization-Depolymerization of Tobacco Mosaic Virus Protein: I. Kinetics

It was shown that a reversible endothermic association of TMV protein subunits (A protein) can take place at pH values below the isoelectric point as well as at pH 6.5. The polymerization occurring below the isoelectric point was found to be more complex than that at pH 6.5 probably because products other than the usual TMV-like rods were formed in addition to those rods and also because side-to-side aggregation of the rods took place readily. Kinetic studies indicated that polymerization can be treated as a second-order linear condensation. The rate of polymerization was found to be a critical function of pH, having a maximum value near pH 4.3. This behavior is at variance with the hypothesis that hydrogen-bonded carboxyl pairs play a dominant rate-determining role in the association of subunits. The dependence of the rate on pH was interpreted to indicate that electrostatic forces between subunits are a significant controlling factor in the polymerization of TMV protein.     

Proteolytic Cleavage of Subunits of the Nucleocapsid of the Paramyxovirus Simian Virus 5

The protein subunits of the nucleocapsid of the parainfluenza virus simian virus 5 isolated from infected cells after dispersion with trypsin, chymotrypsin, or ficin are cleaved proteolytically. The molecular weights of the subunits which result from cleavage depend on the enzyme used, but are around 43,000, compared to the native subunit of 61,000. In most instances cleavage of the subunit appears to be due to the protease used to disperse the cell, and follows cell disruption. Nucleocapsids composed of native, uncleaved subunits can frequently be obtained from infected cells dispersed without a proteolytic enzyme; however, cleavage occasionally occurs even under those conditions, indicating that cellular proteases can at times cleave this protein. Nucleocapsids containing uncleaved subunits can be isolated from cells persistently infected with simian virus 5, indicating that persistent infection is not invariably associated with intracellular cleavage of this protein. Nucleocapsids composed of native subunits are hydrophobic, whereas those composed of the cleaved subunit can be dispersed in aqueous solution. It is suggested that the portion of the molecule removed by cleavage may be responsible for a specific interaction during virus assembly between the nucleocapsid and those areas of plasma membrane which contain the non-glycosylated viral membrane protein, which is also hydrophobic. An amino acid analysis of native and cleaved subunits has been done. The portion of the subunit removed by cleavage does not have a high proportion of hydrophobic residues, suggesting that those present are arranged together to form a hydrophobic domain.The N termini of both the native and cleaved subunits are blocked. This suggests that the portion of the molecule which is externally disposed and removed by cleavage contains the C terminus, and the cleaved subunit which reacts with the viral RNA contains the N terminus.     

Kinetic analysis of the coordinated interaction of SgrAI restriction endonuclease with different DNA targets

SgrAI restriction endonuclease cooperatively interacts and cleaves two target sites that include both the canonical sites, CPuCCGGPyG, and the secondary sites, CPuCCGGPy(A/T/C). It has been observed that the cleaved canonical sites stimulate SgrAI cleavage at the secondary sites. Equilibrium binding studies show that SgrAI binds to its canonical sites with a high affinity (Ka = 4-8 x 10(10) M-1) and that it has a 15-fold lower affinity for the cleaved canonical sites and a 30-fold lower affinity for the secondary sites. Steady-state kinetics reveals substrate cooperativity for SgrAI cleavage on both canonical and secondary sites. The specificity of SgrAI for the secondary site CACCGGCT, as measured by kcat/K is about 500-fold lower than that for the canonical site CACCGGCG, but this difference is reduced to 10-fold in the presence of the cleaved canonical sites. The efficiency of canonical site cleavage also increases by 3-fold when the cleaved canonical sites are present in the reaction. Furthermore, the substrate cooperativity for SgrAI cleavage is abolished for both types of sites in the presence of cleaved canonical sites. These results indicate that target site cleavage occurs via a coordinated interaction of two SgrAI protein subunits, where the subunit bound to the cleaved site stimulates the cleavage of the uncut site bound by the other subunit. The free subunits of SgrAI have the flexibility to bind different target sites and, consequently, assemble into various catalytically active complexes, which differ in their catalytic efficiencies.     

Processing and assembly in vitro of engineered soybean beta-conglycinin subunits with the asparagine-glycine proteolytic cleavage site of 11S globulins

A short interdomain sequence between the N- and C-terminal domains of beta-conglycinin, the major 7S seed storage protein of soybean, was selected as a target for insertion of amino acid residues specifically cleaved by an asparaginyl endopeptidase that processes globulins into acidic and basic chains. Modified beta-conglycinin subunits containing the proteolytic cleavage site self-assembled into trimers in vitro at an efficiency similar to that of the unmodified subunit. In contrast to the absence of cleavage of the unmodified subunits, however, the modified beta-conglycinin trimers were processed by purified soybean asparaginyl endopeptidase into two polypeptides, each the size expected for the beta-conglycinin N- and C-terminal domains, respectively. The cleavage did not alter the assembly of mutant beta-conglycinins and the cleaved mutant trimers remained stable to further proteolytic attack. To examine the possibility of coassembly between the cleaved 11S and 7S subunits, in vitro processed mutant beta-conglycinin subunits were mixed with native dissociated 11S globulin preparations. Reassembly at a high ionic condition did not induce the 7S subunits to interact with 11S subunits to form hexameric complexes. Thus, cleavage of 7S globulin subunits into acidic and basic domains may not be sufficient for hexamer assembly to occur. Biotechnological implications of the engineered proteins are discussed.     

Drosophila acetylcholinesterase. Expression of a functional precursor in Xenopus oocytes.

In insects, acetylcholinesterase is mainly found in the central nervous system. It is expressed in the synapse where it hydrolyzes the neurotransmitter acetylcholine. Maturation of this protein involves several post-translational modifications. The precursor polypeptide is cut at three sites; the N-terminal signal peptide is removed, the C-terminal hydrophobic polypeptide is clipped off and replaced by a glycolipid anchor and the resulting peptide is cut into two polypeptides, corresponding to active subunits. Two of these active subunits are associated to form the final active glycosylated protein. We have expressed the protein via microinjection of an expression vector into Xenopus oocyte nuclei. When the complete cDNA is injected, the acetylcholinesterase formed is biochemically similar to the Drosophila-head acetylcholinesterase. However, the hydrophobic C-terminal peptide is not replaced by a glycolipid anchor. As a consequence, the enzyme is no longer externalized, the proteolytic cutting of the main peptide does not occur and a new polymerization form occurs. Although incompletely processed, this protein is enzymatically active. When a cDNA lacking the coding region of the C-terminal hydrophobic peptide is injected, the resulting acetylcholinesterase is hydrophilic, cleaved into two subunits and secreted into the incubation medium free of contaminants.     

Dissecting quasi-equivalence in nonenveloped viruses: membrane disruption is promoted by lytic peptides released from subunit pentamers, not hexamers

Nonenveloped viruses often invade membranes by exposing hydrophobic or amphipathic peptides generated by a proteolytic maturation step that leaves a lytic peptide noncovalently associated with the viral capsid. Since multiple copies of the same protein form many nonenveloped virus capsids, it is unclear if lytic peptides derived from subunits occupying different positions in a quasi-equivalent icosahedral capsid play different roles in host infection. We addressed this question with Nudaurelia capensis omega virus (NωV), an insect RNA virus with an icosahedral capsid formed by protein α, which undergoes autocleavage during maturation, producing the lytic γ peptide. NωV is a unique model because autocatalysis can be precisely initiated in vitro and is sufficiently slow to correlate lytic activity with γ peptide production. Using liposome-based assays, we observed that autocatalysis is essential for the potent membrane disruption caused by NωV. We observed that lytic activity is acquired rapidly during the maturation program, reaching 100% activity with less than 50% of the subunits cleaved. Previous time-resolved structural studies of partially mature NωV particles showed that, during this time frame, γ peptides derived from the pentamer subunits are produced and are organized in a vertical helical bundle that is projected toward the particle surface, while identical polypeptides in quasi-equivalent subunits are produced later or are in positions inappropriate for release. Our functional data provide experimental support for the hypothesis that pentamers containing a central helical bundle, observed in different nonenveloped virus families, are a specialized lytic motif.     

Structure-function relationships of the Escherichia coli ATP synthase probed by trypsin digestion

Trypsin cleavage has been used to probe structure-function relationships of the Escherichia coli ATP synthase (ECF1F0). Trypsin cleaved all five subunits, alpha, beta, gamma, delta, and epsilon, in isolated ECF1. Cleavage of the alpha subunit involved the removal of the N-terminal 15 residues, the beta subunit was cleaved near the C-terminus, the gamma subunit was cleaved near Ser202, and the delta and epsilon subunits appeared to be cleaved at several sites to yield small peptide fragments. Trypsin cleavage of ECF1 enhanced the ATPase activity between 6- and 8-fold in different preparations, in a time course that followed the cleavage of the epsilon subunit. This removal of the epsilon subunit increased multisite ATPase activity but not unisite ATPase activity, showing that the inhibitory role of the epsilon subunit is due to an effect on cooperativity. The detergent lauryldimethylamine oxide was found to increase multisite catalysis and also increase unisite catalysis more than 2-fold. Prolonged trypsin cleavage left a highly active ATPase containing only the alpha and beta subunits along with two fragments of the gamma subunit. All of the subunits of ECF1 were cleaved by trypsin in preparations of ECF1F0 at the same sites as in isolated ECF1. Two subunits, the beta and epsilon subunits, were cleaved at the same rate in ECF1F0 as in ECF1 alone. The alpha, gamma, and delta subunits were cleaved significantly more slowly in ECF1F0.(ABSTRACT TRUNCATED AT 250 WORDS)     

Subtilisin cleavage of tubulin heterodimers and polymers.

Native pig brain tubulin in heterodimer or polymer form was subjected to limited proteolysis by subtilisin, which is known to cleave at accessible sites within the last 50 amino acids of the highly variable carboxyl-termini of the alpha and beta subunits. Heterodimeric tubulin or tubulin polymerized in the presence of 4 M glycerol or taxol was used in these experiments. Digested tubulin was purified by cycles of polymerization and depolymerization, ammonium sulfate precipitation, or ion-exchange chromatography in the absence or presence of nonionic detergent; however, smaller cleaved products of about 34,000 to 40,000 MW remained associated with the major cleaved subunits, alpha' and beta', under all purification conditions. In order to determine the effect of subtilisin cleavage on tubulin heterogeneity, purified native or subtilisin-cleaved tubulin was subjected to isoelectric focusing, followed by SDS-PAGE. The total number of isotypes was reduced from 17-22 for native alpha,beta tubulin to 7-9 for subtilisin-cleaved alpha',beta' tubulin. When tubulin heterodimers were cleaved, a single major beta' isotype was evident; however, when tubulin polymerized in 4 M glycerol was cleaved, two major beta' isotypes were found. Monoclonal antibodies that recognize a beta carboxyl-terminal peptide, residues 410-430, reacted with both major beta' isotypes, indicating that subtilisin cleavage occurred within the last 20 of the 450 amino acids. In order to establish whether this difference was in fact associated with polymer or heterodimer forms of tubulin, digestion was carried out in the presence of taxol, which stabilizes tubulin polymers. A single major beta' isotype different from the cleaved heterodimer, but coincident with one of the bands of the cleaved glycerol-induced polymers, was found when taxol-treated tubulin was digested. This result suggests the presence of more than one subtilisin site in the beta subunit, near residues 430-435, with different accessibility to the enzyme in the heterodimer and polymer form.     

Tubulin, hybrid dimers, and tubulin S. Stepwise charge reduction and polymerization

Limited proteolysis of rat brain tubulin (alpha beta) by subtilisin cleaves a 1-2-kDa fragment from the carboxyl-terminal ends of both the alpha and beta subunits with a corresponding loss in negative charge of the proteins. The beta subunit is split much more rapidly (and exclusively at 5 degrees C), yielding a protein with cleaved beta and intact alpha subunit, called alpha beta s, which is of intermediate charge. Further proteolysis cleaves the carboxyl terminus of the alpha subunit leading, irreversibly, to the doubly cleaved product, named tubulin S, with a composition alpha s beta s. Both cleavage products are polymerization-competent and their polymers are resistant to 1 mM Ca2+- and 0.24 M NaCl-induced depolymerization. The two polymers differ in that the alpha beta s polymer is stable to cold, GDP, and podophyllotoxin, whereas tubulin S polymer is disassembled by these agents; moreover, alpha beta s forms ring-shaped polymers, whereas alpha s beta s forms filaments associated into bundles and sheets. Tubulin S co-polymerizes with native tubulin yielding a mixed product of intermediate stability. The presence of low mole fractions of tubulin S leads to a marked reduction in the critical concentration for polymerization of the mixture.     

The ribonuclease activity of the cytotoxin alpha-sarcin. The characteristics of the enzymatic activity of alpha-sarcin with ribosomes and ribonucleic acids as substrates

The ribonuclease activity of the cytotoxic protein alpha-sarcin has been characterized. When rat liver ribosomes or 60 S ribosomal subunits were the substrates, alpha-sarcin cleaved a single oligonucleotide of about 488 residues, the alpha-fragment, from the 3' end of 28 S rRNA. In contrast, 40 S ribosomal subunits were not affected by alpha-sarcin. The alpha-fragment was cleaved from 28 S rRNA in 80 S ribosomes when the concentration of alpha-sarcin was 3 x 10(-8) M and the toxin retained its specificity even when the concentration was 3 x 10(-5) M. The turnover number (kcat) for the reaction of alpha-sarcin with ribosomes was 55 min-1, establishing that the toxin acts catalytically. When total rRNA or 28 S rRNA was the substrate, alpha-sarcin caused extensive progressive digestion of the nucleic acids; however, no formation of the alpha-fragment occurred. The extent of the digestion of 28 S rRNA was related to the concentration of alpha-sarcin, but the amount of the toxin required was somewhat greater than that needed with ribosomes. Digestion of homopolynucleotides with alpha-sarcin indicated that the protein is specific for purines. When [32P]5 S rRNA was the substrate, alpha-sarcin cleaved on the 3' side of purines in both single- and double-stranded regions of the molecule. The results suggest that the unusual specificity of alpha-sarcin, in that it cleaves only one of more than 7000 phosphodiester bonds in the ribosome, is a property both of the cytotoxin and of the ribosome.     

Structural changes during the transformation of bacteriophage T4 polyheads: II. Evidence that a conformational change in the major capsid protein precedes the expansion

The shell of the bacteriophage T4 prehead is transformed after the maturation cleavages from a fragile to a highly chemically resistant structure. A “cleaved but anchored” shell, in which the capsid protein has been cleaved but expansion to the mature structure has not yet occurred, is thought to be an intermediate in the transformation. We have compared native, trypsinized, temperature-sensitive mutant, and cleaved but anchored polyheads for differences and similarities in their capsomeres. Our results show that the altered capsomeres of the cleaved but anchored state must be attributed to a conformational change in the subunits, and not simply to the loss of the amino-terminal peptide by proteolysis.     

Assembly properties of fluorescein-labeled tubulin in vitro before and after fluorescence bleaching

Brain tubulin has been conjugated with dichlorotriazinyl- aminofluorescein (DTAF) to form a visualizable complex for the study of tubulin dynamics in living cells. By using several assays we confirm the finding of Keith et al. (Keith, C. H., J. R. Feramisco, and M. Shelanski, 1981, J. Cell Biol., 88:234-240) that DTAF-tubulin polymerizes like control tubulin in vitro. The fluorescein moiety of the complex is readily bleached by the 488-nm line from an argon ion laser. When irradiations are performed over short times (less than 1 s) and in the presence of 2 mM glutathione, a mixture of DTAF-tubulin and control protein (as occurs after microinjection of the fluorescent conjugate into living cells) will retain full polymerization activity. Slow bleaching (approximately 5 min) or bleaching without glutathione promotes formation of covalent cross-links between neighboring polypeptides and kills the polymerization activity of DTAF-tubulin, including some molecules that are neither cross-linked nor bleached. Even under conditions that damage DTAF-tubulin, however, DTAF- microtubules are not destroyed by bleaching. They will continue to elongate by addition of DTAF-tubulin subunits to their free ends, and they neither bind nor exchange subunits along their lateral surfaces. These results suggest that DTAF-tubulin is a suitable analog for tubulin, both in studies of protein incorporation and for investigations of fluorescence redistribution after photobleaching.     

Difference in polymerization and steady-state dynamics of free and gelsolin-capped filaments formed by alpha- and beta-isoactins

The polymerization of scallop β-like actin is significantly slower than that of skeletal muscle α-actin. To reveal which steps of polymerization contribute to this difference, we estimated the efficiency of nucleation of the two actins, the rates of filament elongation at spontaneous and gelsolin-nucleated polymerization and the turnover rates of the filament subunits at steady-state. Scallop actin nucleated nearly twice less efficient than rabbit actin. In actin filaments with free ends, when dynamics at the barbed ends overrides that at the pointed ends, the relative association rate constants of α- and β-actin were similar, whereas the relative dissociation rate constant of β-ATP-actin subunits was 2- to 3-fold higher than that of α-actin. The 2- to 3-fold faster polymerization of skeletal muscle versus scallop Ca-actin was preserved with gelsolin-capped actin filaments when only polymerization at the pointed end is possible. With gelsolin-induced polymerization, the rate constants of dissociation of ATP-actin subunits from the pointed ends were similar, while the association rate constant of β-actin to the pointed filament ends was twice lower than that of α-actin. This difference may be of physiological relevance for functional intracellular sorting of actin isoforms.     

The role of envelope glycoprotein processing in murine leukemia virus infection.

The murine leukemia virus envelope protein is synthesized as a precursor molecule, Pr85env, which is proteolytically cleaved at an arginine residue to produce two mature envelope proteins, gp70 and p15(E). The results presented here indicate that mutation to lysine of the arginine found at the envelope precursor cleavage site results in a precursor which is cleaved with an efficiency at least 10-fold lower than the efficiency with which the wild-type protein is cleaved. This mutation has been used to investigate the requirement for envelope protein processing in various aspects of retroviral infection. Viruses produced by cells transfected with mutant proviral clones are approximately 10-fold less infectious than wild-type viruses. Mutant viruses are incapable of inducing XC cell syncytium formation and are 100-fold less efficient than wild-type viruses at rendering cells resistant to superinfection. Envelope glycoproteins bearing the lysine mutation are found in reduced amounts on the surface of infected cells, and as a result mutant virions contain significantly less envelope protein than do wild-type virions. The phenotypic effects of the processing mutation described here are most likely the result of this paucity of envelope glycoproteins in virions carrying the mutation.     

In vitro polymerization of microtubules from HeLa cells

Although the purification of microtubules from brain by alternate cycles of polymerization and depolymerization in vitro has become routine, the application of this method to non-neural cultured cells has been less successful. Previous investigations have suggested that it was necessary to use substrate-grown cells and 4 M glycerol to obtain microtubules from cultured cells. We have developed a method for preparing microtubules from HeLa cells in spinner cultures without the use of glycerol. Microtubules can be readily carried through two complete cycles of polymerization at 37 degrees C and depolymerization at 4 degrees C in vitro. The microtubules obtained are morphologically similar to brain microtubules in electron micrographs, and the tubulin subunits have mobilities similar to those of brain tubulins on polyacrylamide gels. Typical yields in the second polymerization pellet are about 1 mg protein/ml of packed cells or 2.5-3.0% of the total protein in the soluble cell extract. The major nontubulin protein present after two cycles of polymerization and depolymerization has an apparent mol wt of 68,000 daltons. If glycerol is used during polymerization, this band is virtually absent.     

Scaffolding protein regulates the polymerization of P22 coat subunits into icosahedral shells in vitro

Coat and scaffolding subunits derived from P22 procapsids have been purified in forms that co-assemble rapidly and efficiently into icosahedral shells in vitro under native conditions. The half-time for this reaction is approximately five minutes at 21 degrees C. The in vitro reaction exhibits the regulated features observed in vivo. Neither coat nor scaffolding subunits alone self-assemble into large structures. Upon mixing the subunits together they polymerize into procapsid-like shells with the in vivo coat and scaffolding protein composition. The subunits in the purified coat protein preparations are monomeric. The scaffolding subunits appear to be monomeric or dimeric. These results confirm that P22 procapsid formation does not proceed through the assembly of a core of scaffolding, which then organizes the coat, but requires copolymerization of coat and scaffolding. To explore the mechanisms of the control of polymerization, shell assembly was examined as a function of the input ratio of scaffolding to coat subunits. The results indicated that scaffolding protein was required for both initiation of shell assembly and continued polymerization. Though procapsids produced in vivo contain about 300 molecules of scaffolding, shells with fewer subunits could be assembled down to a lower limit of about 140 scaffolding subunits per shell. The overall results of these experiments indicate that coat and scaffolding subunits must interact in both the initiation and the growth phases of shell assembly. However, it remains unclear whether during growth the coat and scaffolding subunits form a mixed oligomer prior to adding to the shell or whether this occurs at the growing edge.     

Proteolysis of Paracoccus denitrificans cytochrome oxidase by trypsin and chymotrypsin

Paracoccus oxidase containing only two subunits was subjected to proteolysis by trypsin and chymotrypsin. Both subunits of the purified enzyme were cleaved at only a few sites and enzymatic activity was not inhibited. The cleavage sites were identified by protein sequencing. Subunit I was cleaved near the amino-terminus and subunit II in the loop connecting the two predicted trans-membrane helices. In native membrane fragments, but not in intact spheroplasts, this loop was accessible to both proteases. These results provide experimental evidence for the folding of subunit II in the membrane.     

Expression of Torpedo nicotinic acetylcholine receptor subunits in yeast is enhanced by use of yeast signal sequences

We have produced the four subunits of the nicotinic acetylcholine receptor of Torpedo californica, an integral membrane protein, in the yeast Saccharomyces cerevisiae. Two of the subunits (alpha and delta) were readily produced from their cDNAs after simply subcloning them into a yeast shuttle vector adjacent to a yeast promoter. The other two protein subunits (beta and gamma) were not produced by this strategy, although the amounts of mRNA produced from these expression constructs are similar to those for alpha and delta. Replacing the DNA coding for the normal N-terminal signal sequences for the beta and gamma subunits with DNA coding for the signal sequence of yeast invertase results in successful protein synthesis. The yeast signal sequence allows these subunits to be translocated across the membrane of the endoplasmic reticulum and to be glycosylated. The appropriate final size of the subunit proteins suggests that the yeast signal sequence has been properly cleaved after translocation.