Ligand-binding characteristics of recombinant amino- and carboxyl-terminal fragments of human insulin-like growth factor-binding protein-3.

Insulin-like growth factor-binding protein-3 (IGFBP-3) is a member of a family of structurally conserved proteins (IGFBP-1 to -6) which act as carriers and regulators of the mitogenic peptide hormones IGF-I and IGF-II. Members of the IGFBP family share conserved cysteine-rich amino- and carboxyl-terminal regions. The amino-terminal domain of these proteins is recognised to contain an IGF-binding determinant, but evidence to support a binding site in the carboxyl-terminal region of the protein is less rigorous. To further investigate this, we have synthesised both the amino-terminal (residues 1-88; N-88) and carboxyl-terminal (residues 165-264; C-165) domains of human IGFBP-3 in bacteria, as fusion proteins with a carboxyl-terminal FLAG peptide. Although only C-165 showed binding to IGF-I and -II by solution-binding assays, both N-88 and C-165 demonstrated binding to IGF-I and -II by biosensor analysis albeit with reduced affinities compared with full-length IGFBP-3. Only the carboxyl-terminal fragment (C-165) was able to form hetero-trimeric complexes with IGF-I and the acid-labile subunit (ALS). We conclude that the carboxyl-terminal domain of IGFBP-3 contains an IGF-binding determinant and can form ternary complexes with ALS.

Production of recombinant hydroxylated human type III collagen fragment in Saccharomyces cerevisiae.

A recombinant hydroxylated fragment of human type III collagen has been produced in Saccharomyces cerevisiae by coordinated coexpression of a collagen gene fragment together with both the alpha- and beta-subunit genes for prolyl-4-hydroxylase (EC 1.14.11.2). The collagen fragment consisted of 255 residues of the helical domain and the complete C-telopeptide and C-propeptide domains. It was inserted under the control of the ethanol-inducible ADH2 promoter in a multicopy, TRP1-selectable, yeast expression vector, YEpFlag1. The prolyihydroxylase subunit genes were cloned on either side of a bidirectional galactose-inducible promoter in a low-copy minichromosome yeast expression vector, pYEUra3, which is URA3 selectable. Coordinated expression of the three different gene products after cotransformation into S. cerevisiae was detected by immunoblotting. Amino acid analysis of an immunoreactive collagen fraction demonstrated the presence of hydroxyproline, while the presence of a triple-helical domain in the collagen fragment was demonstrated by its resistance to pepsin proteolysis.

Structure-function analysis of human IFN-alpha. Mapping of a conformational epitope by homologue scanning.

The purpose of this study was to map a conformational epitope of a mAb that binds the IFN subtype alpha 4a. Binding of this mAb, designated I-4-A, to IFN-alpha 4a does not block receptor binding, but does neutralize biologic activity by inhibition of signal transduction. A novel strategy was developed, termed homologue scanning, which uses template-coupled polymerase chain reaction to generate hybrid molecules consisting of part (N-terminus) of the reactive IFN-alpha 4a subtype to locate the epitope, and the remainder of the nonreactive IFN-alpha 14 subtype to provide the overall conformation of an IFN-alpha molecule. Hybrid molecules were expressed as 35S-methionine-labeled proteins and tested for immunoreactivity by Western blotting and antiviral activity by cytopathic effect reduction bioassays. Unless an entire IFN-alpha (hybrid) molecule was formed, immunoreactivity and biologic activity were lost, indicating the importance of the C-terminus for correct folding of IFN-alpha molecules. The epitope for I-4-A was localized to the N-terminal 23 residues of IFN-alpha 4a. Furthermore, the immunoreactivity of IFN-alpha 4a analogues, with alterations in the putative receptor-binding region of IFN-alpha 4a residues 30 to 40 was unaffected, in contrast to the biologic activity that was reduced by several orders of magnitude. Thus, the N-terminal 23 residues of IFN-alpha 4a, which probably are not involved in receptor binding, may be important for other interactions of the receptor-bound ligand. In general terms, the novel approach of homologue scanning, using template-coupled PCR to facilitate the generation of hybrid proteins, will have broad application in the mapping of conformational epitopes of proteins that are members of a homologous family. The ability to identify conformational epitopes will increase our understanding important interactions of proteins with antibodies, receptors, and other macromolecules.

Each of three positively-charged amino acids in the C-terminal region of yeast mitochondrial ATP synthase subunit 8 is required for assembly.

Each of three conserved positively-charged residues in the C-terminal region of subunit 8 of yeast (Saccharomyces cerevisiae) mitochondrial ATP synthase was replaced with isoleucine. The assembly and functional properties of the resulting variants (substituted at Arg-37, Arg-42 and Lys-47) were examined using in-vitro systems to assay import into isolated mitochondria and to monitor assembly into ATP synthase, as well as an in-vivo rescue system using host yeast cells lacking endogenous subunit 8. Each such variant was found to be impaired in assembly in vitro, after import in the form of a chimaeric protein bearing a leader sequence with mitochondrial targeting function. Import precursors bearing a duplicated-leader sequence, engendering enhanced delivery to mitochondria of the passenger variant subunit-8 proteins, enabled assembly of the (Lys-47-->Ile) variant to be detected in vitro but not that of (Arg-37-->Ile) or (Arg-42-->Ile) variants. The respiratory growth of subunit 8-deficient host cells could be rescued with the (Lys-47-->Ile) variant expressed allotopically in the nucleus. Such rescued cells were found to have an enhanced growth rate (comparable to that produced by non-mutagenized parental subunit 8) when delivered to mitochondria with the duplicated-leader sequence, as compared to the single-leader sequence. This confirms that the impediment in the (Lys-47-->Ile) variant lies in the efficiency of its assembly, rather than a functional defect, as such, arising from the loss of that positive charge. In contrast, host cells were unable to be rescued by the (Arg-37-->Ile) and (Arg-42-->Ile) variants, even when they were endowed with the duplicated leader sequence. It is concluded that the positively-charged C-terminal domain of subunit 8, common to fungal and mammalian homologues of this protein, plays a key role in its assembly into mitochondrial ATP synthase.

Duplication of secretion signal sequences is deleterious for the secretion of human interferon alpha 4 from Saccharomyces cerevisiae and Bacillus subtilis.

Tandem duplication of a mitochondrial import leader sequence has been shown to markedly increase the efficiency of translocation of chimaeric precursors across mitochondrial membranes to the mitochondrial matrix. The principle of leader sequence duplication was applied to the protein secretion system of the yeast Saccharomyces cerevisiae and of Bacillus subtilis. The secretion signal sequences of yeast invertase and B. subtilis neutral protease were used to direct the secretion of human interferon alpha 4. Our results show that the duplication of these N-terminal signal sequences does not enhance secretion of interferon alpha 4 in either of the cell systems studied.

Structure/function analysis of yeast mitochondrial ATP synthase subunit 8.

Subunit 8 of yeast mitochondrial ATP synthase is a small hydrophobic component of the membrane-associated F0 sector. Structure/function relations in subunit 8 were studied by focusing on three structural domains: a highly conserved NH2-terminal region, a central hydrophobic region (previously suggested to be a transmembrane stem), and a COOH-terminal region bearing a conserved array of three positively charged residues. A combined approach was used, which encompasses site-directed mutagenesis, in vitro import and assembly tests, and an in vivo allotopic expression system (using host cells unable to synthesise subunit 8 in mitochondria). The results indicate that the NH2-terminal region of subunit 8 is involved functionally in the F0 sector. As the central hydrophobic region can functionally tolerate the introduction of multiple, positively charged residues (which abolishes the proteolipid solubility characteristics of the entire subunit), the role of this hydrophobic region as a transmembrane stem is brought into question. Each of the three positively charged residues toward the COOH-terminus of subunit 8 is required for the efficient assembly of this subunit into the F0 sector. Removal of the more proximal charged residues Arg37 or Arg42 has a more severe impact on subunit 8 assembly than does removal of the most distal residue Lys47 in terms of both in vitro import and assembly as well as the ability of the subunit 8 variant to function in mitochondrial ATP synthase in vivo.

Duplication of leader sequence for protein targeting to mitochondria leads to increased import efficiency.

We describe a novel method for enhancing protein import into mitochondria, by tandemly duplicating the N-terminal cleavable leader peptide using a gene manipulation strategy. The import into isolated yeast mitochondria of passenger proteins (yeast mitochondrial ATP synthase subunits 8 and 9 and some mutagenised derivatives) that show little or no import when endowed with one such leader (that of Neurospora crassa mitochondrial ATP synthase subunit 9) is remarkably improved when the leader is tandemly duplicated. The import of these chimaeric proteins bearing a double leader is so rapid that a series of partially processed precursor intermediates accumulates inside the mitochondria before the final proteolytic release of leader sequences from the passenger proteins. It is considered that the duplicated leader greatly accelerates delivery of the import precursors to outer membrane receptor elements and the associated translocation systems, thereby enhancing precursor uptake into mitochondria.

Aberrant mitochondrial processing of chimaeric import precursors containing subunits 8 and 9 of yeast mitochondrial ATP synthase.

A set of chimaeric precursors which contain the same leader sequences but different passenger proteins has been analyzed for the site of protease cleavage following import into yeast mitochondria. Each precursor comprises the leader of Neurospora crassa subunit 9 of mitochondrial ATP synthase fused to subunit 8 or 9 of the corresponding yeast enzyme. Precursors containing the first five residues of mature N. crassa subunit 9 interposed between the leader and the yeast passenger protein were cleaved at the natural site of the N. crassa subunit 9 precursor. Direct fusions without interposed sequences were cleaved at novel sites. Cleavage occurred between the 3rd and 4th residues of yeast subunit 8, but for yeast subunit 9, cleavage occurred within the leader, 8 residues upstream of the passenger protein.

Amino acid substitutions in mitochondrial ATP synthase subunit 9 of Saccharomyces cerevisiae leading to venturicidin or ossamycin resistance.

A series of mitochondrially inherited mutants of yeast has been analysed, which were previously identified as showing resistance to the antibiotics venturicidin or ossamycin and whose mutations showed tight linkage to oligomycin-resistance alleles affecting subunit 9 of the mitochondrial ATP synthase. DNA sequence analysis of the oli1 gene of these mutants has been used to define the nature of amino acid substitution in the subunit 9 protein. In the case of the two venturicidin-resistant mutants, mutations affect amino acids on the N-terminal stem of the protein, namely Gly25----Ser (venR ossS oliR) and Ala 27----Gly (venR ossS oliS). The mutations found in the two ossamycin-resistant mutants affect amino acids on the C-terminal stem of the protein; namely Leu53----Phe (vanS ossR oliR) and Leu57----Phe (venS ossR oliS). These results allow us to further develop a fine structure map of domains within the subunit 9 protein involved in antibiotic interaction.