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.

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 aminoand 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 solutionbinding 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 carboxylterminal domain of IGFBP-3 contains an IGF-binding determinant and can form ternary complexes with ALS.

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Wc descrtb~ a novel method for enhancinj protein import into mltochondria, by [andemly dup!icating the N.termi~al elc~tvabl© leader l~pttde usinll a llcnc manipuhttlon strategy. The import into i~olated >'east mitoehondria of On,senator proteins (yeast mitochondrial ATE syntha~ ~,~Jbunits 8 and 9 anti some mutalienised derivatives) that show little or no import when endowed with one such leader (that of Neuraspura cra#~a mitochon. drial ATE ~ynzhase subunit 91 is remarkably improved wt~en the leader is randomly duplicated. The import or these ehimaeric proteins bc~rin~ a double Io=tder is so rapid that a series of partially processed precursor intem~ediates accumulates inside the mitochondria b~fore the final prot¢olyt-ie release of leader sequences from the passent!er proteins. It is considered that the duplicated leader Breatly accelerates delivery of the import precur.sors to outer membrane receptor dements ~tnd the associated translocation systcnls, thereby enhancing precursor uptake into mitochondria, Mitoehondrlal ATPase complex; Chimacrie fusion protein: Integral membrane protein: Leader peptide; Protein import: Saecharomyee# ccrevl~ic~¢

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 olil 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 venturicidinresistant mutants, mutations affect amino acids on the N-terminal stem of the protein, namely Gly25~Ser (ven Ross s oli R) and Ala 27~Gly (ven Ross s oliS). The mutations found in the two ossamycin-resistant mutants affect amino acids on the C-terminal stem of the protein, namely Leu53--*Phe (van s oss R oli R) and Leu57--*Phe (ven s oss R oliS). These results allow us to further develop a fine structure map of domains within the subunit 9 protein involved in antibiotic interaction.