N-fatty acyl compounds inhibit myristoyl acylation of pp60v-src and reduce tumorigenicity of Rous sarcoma virus-infected cells.

Prevention of NH2-terminal myristoylation of pp60v-src was determined with N-fatty acyl glycinal derivatives. Of all the compounds tested, N-myristoyl and N-lauroyl glycinal diethylacetal, N-myristoyl glycyl glycinal diethylacetal, and N-myristoyl-4-aminobutyl aldehyde diethylacetal strongly inhibited myristoylation of pp60v-src; but N-myristoyl diglycyl, N-myristoyl triglycyl, N-decanoyl glycinal diethylacetal, and N-palmitoyl glycinal diethylacetal did not. N-Myristoyl glycinal diethylacetal (25 or 50 microM) suppressed both morphological transformation and colony formation of Rous sarcoma virus-infected chick embryo fibroblasts.     

N-myristoyl transferase assay using phosphocellulose paper binding.

N-myristoyl-CoA:protein N-myristoyl transferase is the enzyme that catalyzes the covalent transfer of myristic acid to the NH2-terminal glycine residue of a protein, or peptide, substrate. We have established a new, rapid, reliable, and inexpensive myristoyl-CoA:protein N-myristoyl transferase assay. This N-myristoyl transferase assay is based on the binding of the [3H]myristoylated peptide to a P81 phosphocellulose paper matrix and is more convenient for assaying multiple samples than existing procedures. Two peptides, derived from the N-terminal sequences of the type II catalytic subunit of cAMP-dependent protein kinase and pp60src, were used as substrates. A survey of rat and bovine tissue extracts demonstrated that in both cases brain contained the highest NMT activity (i.e., brain greater than spleen greater than heart greater than liver). Under the assay conditions used, the rate of myristoylation was linear for 10 min and with up to 4.0 mg/ml of brain extract.     

Antibodies to an NH2-terminal myristoyl glycine moiety can detect NH2-terminal myristoylated proteins in the retrovirus-infected cells

Novel antibodies were raised against a synthetic NH2-terminal myristoyl glycine moiety which is characteristic of N-myristoyl-proteins. Antisera raised against N-myristoyl-Gly-hemocyanin reacted with N-myristoyl-Gly-[125I]albumin. The immunoreaction was competed for by albumin conjugated with N-myristoyl-glycine, while underivatized albumin had no effect. Of the [3H]myristate-labeled proteins detected, pp60v-src, which is a transforming protein of Rous sarcoma virus, and p19gag and p17gag, which are core proteins in the human T-cell leukemia virus and the human immunodeficiency virus, were identified as N-myristoylated proteins by the radioimmunoprecipitation analyses with the antibody.     

The ADP/ATP carrier is the 32-kilodalton receptor for an NH2-terminally myristylated src peptide but not for pp60src polypeptide.

Membrane binding of pp60src is initiated via its myristylated NH2 terminus. To identify a candidate pp60src docking protein or receptor in the membrane, a radiolabelled peptide corresponding to the pp60src NH2-terminal membrane binding domain was cross-linked to fibroblast membranes and found to specifically label a 32-kDa protein. This protein was purified by appending an affinity tag to the peptide probe so that the cross-linked complex could be isolated via affinity chromatography. Microsequencing indicated that the 32-kDa protein was the mitochondrial ADP/ATP carrier (AAC). This result was further confirmed by the ability of an antibody to the AAC to immunoprecipitate the cross-linked complex, by the ability of certain inhibitors of the AAC to block cross-linking, and by membrane fractionation to show that complex formation occurred essentially exclusively in the mitochondrial fraction. While the AAC bound the myristyl-src peptide in a specific manner both in vitro and in vivo, its localization to the inner membrane of the mitochondrion precludes its being a pp60src binding protein. An analysis of pp60v-src binding in vitro was consistent with this expectation. Thus, use of a myristyl-src peptide revealed an unexpected and previously unidentified binding capacity of the AAC, most likely related to the ability of long-chain fatty acyl coenzyme As to serve as AAC inhibitors. The amphipathic nature of the pp60src NH2 terminus suggests alternative strategies for uncovering pp60src membrane binding species.     

Characterization of the Rous sarcoma virus transforming gene product

This report describes quantitative results of in vitro phosphorylation of the Rous sarcoma virus transforming gene product, pp60src, using ATP or GTP as phosphate donors. The Km values for the phosphorylation of pp60src by ATP and GTP were similar (10-36 and 25-36 microM, respectively) and the Vmax values were different (5-7 and 1.5-1.7 nmol min-1 mg-1 of pp60src, respectively). The radiolabeling of pp60src by [gamma-32P] ATP was inhibited by ADP and dATP at 20-fold higher concentrations by 75 and 83%, respectively. Other nucleotides served as weaker inhibitors under the same conditions. The radiolabeling of pp60src by [gamma-32P]GTP had lower specificity for this nucleotide, since ATP, dATP, ADP, dGTP, GDP, and TTP had at least a 50% inhibitory effect. The phosphorylated products of approximate Mr = 60,000 that were produced with ATP or GTP were shown to be the same protein molecule since they both could be immunoprecipitated with antibody raised against p60src produced by bacterial recombinants. Structural analysis revealed that the use of GTP resulted in phosphorylation of a tyrosine residue on the COOH-terminal region of pp60src, apparently the same site which contains the tyrosine phosphorylated in infected cells. In contrast, the use of ATP resulted in phosphorylation of several additional tyrosine residues on the NH2-terminal region of the molecule. In thermolability studies, the t1/2 values for the phosphorylation of pp66src in preparations from wild type virus-infected chicken cells were 5.1 min for both ATP and GTP, whereas the t1/2 values for the phosphorylation of pp60src in preparations from temperature-sensitive transformation mutant-infected cells were 1.1 min for both phosphate donors. Similar observations were found with alpha-casein as substrate.     

Antibody as an immunological probe for studying the refolding of bovine serum albumin. An immunochemical approach to the identification of possible nucleation sites.

Specifically purified antibody to either domain I, domain III, or to subregions of domains I or III of serum albumin was added to refolding mixtures containing reduced serum albumin but no other refolding catalyst. It was found that the refolding of reduced albumin was greatly enhanced by the presence of specific antibody in the refolding mixture, that this enhancement was restricted to that domain for which the added antibody was directed, and that antibody-mediated enhancement of refolding in the NH2-terminal portion of each domain was delayed as compared to that seen in the COOH-terminal portion of each domain. Thus, an apparent COOH-terminal to NH2-terminal pathway of refolding within each domain was observed, which is consistent with the proposed evolutionary pathway of the albumin molecule and also consistent with the proposed presence of a nucleation center in the COOH-terminal double disulfide loop of each domain.     

Alterations in pp60c-src accompany differentiation of neurons from rat embryo striatum.

Cultured neurons from rat embryo striatum were found to contain two structurally distinct forms of pp60c-src. The 60-kilodalton (kDa) form appeared similar to pp60c-src from cultured rat fibroblasts or astrocytes. The 61-kDa form was specific to neurons and differed in the NH2-terminal 18 kDa of the molecule. In undifferentiated neurons the predominant phosphorylated species of pp60c-src was the fibroblast form. Upon differentiation, a second phosphorylated form of pp60c-src was detected. This form had two or more additional sites of serine phosphorylation within the NH2-terminal 18-kDa region of the molecule, one of which was Ser-12. The specific protein-tyrosine kinase activity of the total pp60c-src population increased 14-fold, as measured by autophosphorylation, or 7-fold, as measured by phosphorylation of an exogenous substrate, as striatal neurons differentiated. This elevation in protein kinase activity occurred without a detectable decrease in Tyr-527 phosphorylation or increase in Tyr-416 phosphorylation. Our results support the idea that the expression of the neuron-specific form of pp60c-src and the increase in specific protein kinase activity may be important for neuronal differentiation.     

Human cellular src gene product: identification of the myristoylated pp60c-src and blockage of its myristoyl acylation with N-fatty acyl compounds resulted in the suppression of colony formation

A p60K protein in human colon adenocarcinoma tumor cell lines was identified as a myristoylated pp60c-src by fluorography and radioimmunoprecipitation analysis. Prevention of the myristoylation of pp60c-src was determined with N-fatty acyl glycinal compounds. Of the compounds tested, N-myristoyl glycinal diethylacetal, N-lauroyl glycinal diethylacetal, N-myristoyl glycyl glycinal diethylacetal, and N-myristoyl-4-aminobutyl-aldehyde diethylacetal strongly blocked the myristoylation, but N-decanoyl glycinal diethylacetal and N-palmitoyl glycinal diethylacetal did not. The myristoyl blocking compounds depressed colony formation, cell proliferation, and specific localization to the plasma membrane of pp60c-src. The results taken together suggest that myristoylation of the c-src oncogene product may be very important for tumorigenicity of c-src gene expressed cells.     

Isolation and partial characterization of a monoclonal antibody to the Rous sarcoma virus transforming protein pp60src.

Transformation of cells by Rous sarcoma virus is mediated by the product of the viral src gene, pp60src. A hybridoma cell line producing an immunoglobulin G3 antibody to pp60src was isolated after lymph node cells from immune mice were fused with mouse myeloma cells (P3-NS1-1). Mice were immunized with p60src purified from Escherichia coli cells expressing the src gene product. The monoclonal antibody immunoprecipitated pp60src from Rous sarcoma virus-transformed cells and recognized an antigenic determinant located in the amino-terminal third of the pp60src protein.     

Isolation of monoclonal antibodies that recognize the transforming proteins of avian sarcoma viruses.

Thirteen clones of hybrid cells which synthesize antibodies directed against the Rous sarcoma virus (RSV) transforming protein, pp60src, were isolated. Mouse myeloma cells were fused with spleen cells from mice that had been immunized with purified pp60src from bacterial recombinants which direct the synthesis of the RSV src gene. The hybridomas which survived the selection medium were screened by immunoprecipitation of pp60src from 32P-labeled lysates of RSV-transformed cells. Monoclonal antibodies produced by subclones derived from 13 hybridomas recognized pp60src encoded by the Schmidt-Ruppin and Prague strains of RSV and the cellular homolog of pp60src. Antibody from clone 261 had a high affinity for the viral yes gene product, and antibodies from clones 443 and 463 recognized the transforming proteins encoded by viruses containing the related transforming genes fps and ros. Several other clones had a low affinity for the viral yes, fps, and ros gene products which could be detected by in vitro phosphorylation of the transforming proteins after immunoprecipitation with the monoclonal antibody. All of the monoclonal antibodies allowed phosphorylation of pp60src and casein in an immune complex-bound reaction.     

Comparison of huntingtin proteolytic fragments in human lymphoblast cell lines and human brain

Proteolytic fragments of huntingtin (htt) in human lymphoblast cell lines from HD and control cases were compared to those in human HD striatal and cortical brain regions, by western blots with epitope-specific antibodies. HD lymphoblast cell lines were heterozygous and homozygous for the expanded CAG triplet repeat mutations, which represented adult onset and juvenile HD. Lymphoblasts contained NH(2)- and COOH-terminal htt fragments of 20-100 kDa, with many similar htt fragments in HD compared to control lymphoblast cell lines. Detection of htt fragments in a homozygous HD lymphoblast cell line demonstrated proteolysis of mutant htt. It was of interest that adult HD lymphoblasts showed a 63-64 kDa htt fragment detected by the NH(2)-domain antibody, which was not found in controls. In addition, control and HD heterozygous cells showed a common 60-61 kDa band (detected by the NH(2)-domain antibody), which was absent in homozygous HD lymphoblast cells. These results suggest that the 63-64 kDa and 60-61 kDa NH(2)-domain htt fragments may be associated with mutant and normal htt, respectively. In juvenile HD lymphoblasts, the presence of a 66-kDa, instead of the 63-64 kDa N-domain htt fragment, may be consistent with the larger polyglutamine expansion of mutant htt in the juvenile case of HD. Lymphoblasts and striatal or cortical regions from HD brains showed similarities and differences in NH(2)- and COOH-terminal htt fragments. HD striatum showed elevated levels of 50 and 45 kDa NH(2)-terminal htt fragments [detected with anti(1-17) serum] compared to controls. Cortex from HD and control brains showed similar NH(2)-terminal htt fragments of 50, 43, 40, and 20 kDa; lymphoblasts also showed NH(2)-terminal htt fragments of 50, 43, 40, and 20 kDa. In addition, a 48-kDa COOH-terminal htt band was elevated in HD striatum, which was also detected in lymphoblasts. Overall, results demonstrate that mutant and normal htt undergo extensive proteolysis in lymphoblast cell lines, with similarities and differences compared to htt fragments observed in HD striatal and cortical brain regions. These data for in vivo proteolysis of htt are consistent with the observed neurotoxicity of recombinant NH(2)-terminal mutant htt fragments expressed in transgenic mice and in transfected cell lines that may be related to the pathogenesis of HD.     

Antipeptide antibodies define the NH2-terminal structure of the pan-specific hemopoietin interleukin 3

Antipeptide antibodies were raised in rabbits against a synthetic peptide including the six NH2-terminal amino acids of the pan-specific hemopoietin interleukin 3 (IL 3). Affinity-purified antibody preparations specific for epitopes determined by residues 1 to 6 were immobilized and used as affinity columns. Up to 98% of IL 3 bioactivity in T cell-conditioned medium was depleted by these columns, as was 71 and 74%, respectively, of IL 3 aberrantly produced by the myeloid leukemias WEHI-274.14 and WEHI-3B. IL 3 produced in vivo in WEHI-3B tumor-bearing mice also bound to the anti-1-6 antibody column, up to 70% of the bioactivity being depleted from ascites fluid and up to 84% from the serum. These results suggest that all IL 3 secreted by T cells and the majority of the IL 3 molecules secreted by myeloid leukemias express epitopes determined by residues 1-6 and cannot have the NH2-terminal amino acid sequence initially reported for IL 3. These six NH2-terminal amino acids share similarities with the NH2-terminal amino acids of several other lymphokines, suggesting an important function for this hexapeptide.     

Detection and localization of a cytoplasmic domain on the beta-subunit of Na+,K+-ATPase. A monoclonal antibody study

A monoclonal antibody directed against the beta-subunit of dog kidney Na+,K+-ATPase was generated. Immunoblots demonstrate that monoclonal antibody III 18A binds exclusively to the denaturated beta-subunit. Binding experiments with membranes and whole cells reveal that III 18A binds to membranes but not to whole cells, indicating that the antibody binds to a cytoplasmic domain on the native beta-subunit. To localize the antibody-binding epitope, purified membrane-bound enzyme was fragmented by protease treatment. Tryptic digestion yields a 30-kDa fragment of the beta-subunit, which still retains the binding capacity for the antibody. Thus III 18A probably does not bind to the NH2-terminal segment of the protein. On the other hand, fragmentation of the beta-subunit with low concentrations of papain, which is known to yield a 40-kDa NH2-terminal and a 16-kDa COOH-terminal fragment, results in a complete loss of III 18A binding. These results suggest that the antibody-binding epitope is localized at or near a papain cleavage site on the COOH-terminal part of the beta-subunit. This is inconsistent with a structure model of the beta-subunit containing only a single transmembrane hydrophobic segment with a cytoplasmic NH2-terminal portion, but agrees quite well with a hypothetical structure with four intramembrane segments.     

Identification of platelet membrane proteins that interact with amino-terminal peptides of pp60c-src.

Platelets contain exceptionally high levels of pp60c-src and, thus, provide a convenient system for investigating the physiological function of this protein-tyrosine kinase. We have employed chemical cross-linking of myristylated amino-terminal peptides of pp60c-src to platelet membranes in order to identify platelet membrane components that interact with pp60c-src to regulate or mediate its activity. We detected specific binding of radioiodinated peptides to platelet membrane proteins of 32, 50, 92, and 105 kDa. The 32-kDa protein may be related to the putative src receptor component recently identified in fibroblast membranes. The most reactive platelet protein, however, is the 50-kDa protein, which is either absent or nonreactive in fibroblast membranes. Binding of src peptides to this protein was saturable, and we estimate the presence of approximately 1 x 10(6) of the 50-kDa binding sites per platelet. The specificity of the peptide binding to the 50- and 32-kDa platelet proteins was analyzed by competition with different peptides. The binding sites displayed an absolute requirement for an N-myristoyl moiety and a strong preference for pp60c-src amino-terminal sequences. The identification of these src-interacting proteins may help to decipher the biochemical pathways in which platelet pp60c-src is involved.     

Detection of the Viral Sarcoma Gene Product in Cells Infected with Various Strains of Avian Sarcoma Virus and of a Related Protein in Uninfected Chicken Cells

Genetic analyses have defined a single gene (src) as that portion of the avian sarcoma virus (ASV) genome which encodes the protein directly responsible for ASV-induced neoplastic transformation. We have recently identified the polypeptide product of the src gene of the Schmidt-Ruppin (SR) strain of ASV, a 60,000-dalton phosphoprotein designated pp60(src), and have further determined that pp60(src) acts as a protein kinase. Essential to the identification and characterization of the pp60(src) protein of SR-ASV was the use of serum (TBR serum) from rabbits bearing SR-ASV-induced tumors. TBR serum was, however, strain specific, recognizing pp60(src) from SR-ASV-transformed cells only. We report here that sera from marmosets bearing tumors induced by the Bryan or SR strains of ASV (TBM sera) contain antibody which precipitates the transforming gene product from cells transformed by the SR, Bryan, Prague, or Bratislava strains of ASV. In contrast, rabbits bearing tumors induced by either the Bratislava or Bryan strains of ASV, or hamsters with SR-ASV-induced tumors did not produce antibody to pp60(src) from any strain of ASV. The 60,000-dalton polypeptides immunoprecipitated with TBM serum from cells transformed by each of the above virus strains are phosphoproteins. One-dimensional peptide mapping by limited proteolysis revealed that the pp60(src) proteins are structurally very similar, but not identical. Furthermore, all of the viral pp60(src) proteins have an associated phosphotransferase activity. In addition to detecting the viral src proteins, TBM serum was able to immunoprecipitate an antigenically related protein from normal uninfected avian cells.     

Antiserum specific for the carboxy terminus of the transforming protein of Rous sarcoma virus

An antiserum specific for the carboxy terminus of p60src, the transforming protein of Rous sarcoma virus, was produced by immunization of rabbits with a conjugate of bovine serum albumin and the synthetic peptide NH2-Tyr-Val-Leu-Glu-Val-Ala-Glu-COOH. The carboxy-terminal six amino acids of this peptide correspond in sequence to that deduced for the carboxy terminus of the p60src of the Schmidt-Ruppin strain of Rous sarcoma virus of subgroup A. The p60src proteins of the several strains of Rous sarcoma virus and the cellular homolog of the viral transforming protein, p60c-src, comprise a polymorphic family of polypeptides. The anticarboxy-terminal serum reacted readily with the p60src proteins of three different strains of Rous sarcoma virus. In contrast, no precipitation of cellular p60c-src could be detected with this serum. This suggests that the viral p60src proteins have identical carboxy termini and that the carboxy terminus of cellular p60c-src may be different from that of viral p60src. The anticarboxy-terminal serum reacted poorly with the subpopulation of viral p60src which is present in a complex with two cellular phosphoproteins. Apparently, the presence of the two cellular proteins interferes with the recognition of p60src by the anticarboxy-terminal serum. It seems likely, therefore, that these two cellular proteins bind to the carboxy-terminal domain of p60src.     

A monoclonal antibody specific for Lys-plasminogen. Application to the study of the activation pathways of plasminogen in vivo

Human plasminogen, a glycoprotein with NH2-terminal Glu, is rapidly converted by traces of plasmin to proteolytic derivatives with NH2-terminal Met 68, Lys 77, or Val 78 ("Lys-plasminogen"), which are much more readily activated to plasmin than is Glu-plasminogen. It has, therefore, been proposed that physiological activation of Glu-plasminogen occurs mainly via Lys-plasminogen intermediates (Wiman, B., and Wallén, P. (1973) Eur. J. Biochem. 36, 25-31). In the present study we have characterized a murine monoclonal antibody (LPm1) directed against an epitope exposed in Lys-plasminogen but not in Glu-plasminogen. The antibody was secreted by a hybridoma obtained by fusion of mouse myeloma cells (P3X63-Ag8-6.5.3) with spleen cells of a mouse immunized with purified Lys-plasmin-alpha 2-antiplasmin complex. Coupling of the alpha-amino groups of Lys-plasminogen with phenylisothiocyanate resulted in complete loss of immunoreactivity for LPm1, which was, however, fully restored by cleavage of the derivatized NH2-terminal amino acid. After a second cycle, immunoreactivity was not restored, indicating that the LPm1 antibody-binding site depends on the presence of Lys 77 and/or Val 78 as NH2-terminal amino acids. The immunoreactivity of Lys-plasminogen with LPm1 is abolished by reduction of the protein, suggesting that conversion of Glu-plasminogen to Lys-plasminogen is associated with a conformational alteration exposing the epitope for the LPm1 monoclonal antibody. In order to investigate the pathways of plasminogen activation in vivo, total plasmin-alpha 2-antiplasmin and Lys-plasmin-alpha 2-antiplasmin complexes were measured with sandwich-type micro enzyme-linked immunosorbent assays. Therefore, microtiter plates were coated with monoclonal antibodies against alpha 2-antiplasmin, and bound antigen was quantitated with horseradish peroxidase-conjugated LPm1 or a monoclonal antibody reacting equally well with Glu-plasmin as with Lys-plasmin. In 25 healthy subjects the plasmin-alpha 2-antiplasmin levels in plasma were undetectable (less than 0.1 nM). Infusion of tissue-type plasminogen activator in patients with thromboembolic disease resulted in generation of high concentrations of Glu-plasmin-alpha 2-antiplasmin complex (620 +/- 150 nM, n = 7) whereas neither Lys-plasmin-alpha 2-antiplasmin complex nor Lys-plasminogen were consistently detected. It is, therefore, concluded that activation of the fibrinolytic system in vivo occurs by direct cleavage of the Arg 560-Val 561 bond in Glu-plasminogen and not via formation of the Lys-plasminogen intermediates.     

Synthetic peptide substrates for a tyrosine protein kinase

Immunoprecipitates containing the transforming protein of the avian sarcoma virus, Y73, together with its associated tyrosine-specific protein kinase, have an activity which will phosphorylate the synthetic peptide Lys-Leu-Ile-Glu-Asp-Asn-Glu-Tyr-Thr-Ala-Arg at the tyrosine residue. This peptide corresponds to 10 out of 11 amino acids surrounding the phosphorylated tyrosine in both pp60src and P90, the transforming proteins of Rous sarcoma virus and Y73 virus, respectively. The apparent Km for phosphorylation of the peptide was about 5 mM. A second peptide with the sequence Lys-Leu-Ile-Asp-Asn-Glu-Tyr-Thr-ala-Arg differing from the first peptide only by the absence of the glutamic acid 4 residues from the tyrosine was also phosphorylated, but the apparent Km for the reaction was 40 mM. Several sites of tyrosine phosphorylation in viral transforming proteins have been found to have one or more glutamic acids close to the phosphorylated tyrosine on the NH2-terminal side. Taken together with our in vitro phosphorylation studies, this suggests that the primary sequence surrounding target tyrosines may play a role in recognition of substrates by tyrosine protein kinases, and in particular, that glutamic acid residues on the NH2-terminal side may be important.     

The prepro-peptide of Mucor rennin directs the secretion of human growth hormone by Saccharomyces cerevisiae

An aspartic proteinase, Mucor pusillus rennin (MPR), of filamentous fungus Mucor pusillus, is efficiently secreted from a transformant of Saccharomyces cerevisiae containing the intact MPR gene. To test the usefulness of the MPR leader peptide in secretion of heterologous proteins from yeast cells, several plasmids encoding the fusion proteins composed of different parts of the NH2-terminal region of prepro-MPR and human growth hormone (hGH) were constructed. The parts of the leader peptide upstream of hGH were the whole prepro-peptide following the NH2-terminal region of mature MPR in JGH1, the intact pre-sequence and a part of the pro-sequence in JGH2, and the putative signal sequences of the NH2-terminal 18 and 22 amino acids in JGH3 and JGH7, respectively. When the hGH genes fused to these leader sequences were expressed in yeast cells under the control of the yeast GAL7 promoter, proteins of various sizes immunoreactive with the anti-hGH antibody were secreted into the medium. Among the plasmids mentioned above, JGH2 directed the greatest secretion of the protein of 23 kilodaltons in size, which contained the expected NH2-terminal amino acid sequence of an additional eight amino acids derived from the pro-peptide of MPR. The addition of the GAL10 terminator downstream of the hGH gene in JGH2 resulted in a greater than three- to fivefold increase in the secretion, whereas the insertion of the GAL4 gene, which is a positive regulator for the GAL system, had no significant effect. The improved yield of the total protein of hGH secreted into the medium reached approximately 10 mg/liter.     

The prepro-peptide of Mucor rennin directs the secretion of human growth hormone by Saccharomyces cerevisiae.

An aspartic proteinase, Mucor pusillus rennin (MPR), of filamentous fungus Mucor pusillus, is efficiently secreted from a transformant of Saccharomyces cerevisiae containing the intact MPR gene. To test the usefulness of the MPR leader peptide in secretion of heterologous proteins from yeast cells, several plasmids encoding the fusion proteins composed of different parts of the NH2-terminal region of prepro-MPR and human growth hormone (hGH) were constructed. The parts of the leader peptide upstream of hGH were the whole prepro-peptide following the NH2-terminal region of mature MPR in JGH1, the intact pre-sequence and a part of the pro-sequence in JGH2, and the putative signal sequences of the NH2-terminal 18 and 22 amino acids in JGH3 and JGH7, respectively. When the hGH genes fused to these leader sequences were expressed in yeast cells under the control of the yeast GAL7 promoter, proteins of various sizes immunoreactive with the anti-hGH antibody were secreted into the medium. Among the plasmids mentioned above, JGH2 directed the greatest secretion of the protein of 23 kilodaltons in size, which contained the expected NH2-terminal amino acid sequence of an additional eight amino acids derived from the pro-peptide of MPR. The addition of the GAL10 terminator downstream of the hGH gene in JGH2 resulted in a greater than three- to fivefold increase in the secretion, whereas the insertion of the GAL4 gene, which is a positive regulator for the GAL system, had no significant effect. The improved yield of the total protein of hGH secreted into the medium reached approximately 10 mg/liter.