Mitochondrial protein import: identification of processing peptidase and of PEP, a processing enhancing protein

Transport of nuclear-encoded precursor proteins into mitochondria includes proteolytic cleavage of amino-terminal targeting sequences in the mitochondrial matrix. We have isolated the processing activity from Neurospora crassa. The final preparation (enriched ca. 10,000-fold over cell extracts) consists of two proteins, the matrix processing peptidase (MPP, 57 kd) and a processing enhancing protein (PEP, 52 kd). The two components were isolated as monomers. PEP is about 15-fold more abundant in mitochondria than MPP. It is partly associated with the inner membrane, while MPP is soluble in the matrix. MPP alone has a low processing activity whereas PEP alone has no apparent activity. Upon recombining both, full processing activity is restored. Our data indicate that MPP contains the catalytic site and that PEP has an enhancing function. The mitochondrial processing enzyme appears to represent a new type of "signal peptidase," different from the bacterial leader peptidase and the signal peptidase of the endoplasmic reticulum.     

Differential expression of kunitz and bowman-birk soybean proteinase inhibitors in plant and callus tissue

Bowman-Birk soybean trypsin inhibitor (BBSTI) but not Kunitz soybean trypsin inhibitor (KSTI) was found in samples of undifferentiated and partially differentiated Amsoy 71 tissue culture callus. This suggests the differential metabolism of these two classes of proteinase inhibitors, whether the difference be in synthesis, in rates of degradation, or both. The differential metabolism of the proteinase inhibitors is also seen in the plant. Both BBSTI and KSTI were found in the hypocotyl, root, and epicotyl of the Amsoy 71 soybean seedling in addition to their expected presence in the cotyledons. Whereas the ratio of KSTI to BBSTI in the cotyledon was higher, the ratio of BBSTI to KSTI was higher in the extracotyledonary tissues of the seedling. The levels of both classes of proteinase inhibitors declined during seedling growth, except in the epicotyl and the proximal root. In both of these tissues, an increase in BBSTI, but not in KSTI content, expressed as milligrams inhibitor per plant part, occurred.     

Regulation of Newly Evolved Enzymes. I. Selection of a Novel Lactase Regulated by Lactose in ESCHERICHIA COLI

Thirty-four lactose-utilizing strains of E. coli were selected from a lac Z deletion strain. In 31 of these, the synthesis of the newly evolved lactase is regulated by lactose. The lactase activity in all the strains is indistinguishable from the ebg(+) activity identified by Campbell, Lengyel and Langridge (1973).     

cis-active elements from mouse chromosomal DNA suppress simian virus 40 DNA replication.

Simian virus 40 (SV40)-containing DNA was rescued after the fusion of SV40-transformed VLM cells with permissive COS1 monkey cells and cloned, and prototype plasmid clones were characterized. A 2-kilobase mouse DNA fragment fused with the rescued SV40 DNA, and derived from mouse DNA flanking the single insert of SV40 DNA in VLM cells, was sequenced. Insertion of the intact rescued mouse sequence, or two nonoverlapping fragments of it, into wild-type SV40 plasmid DNA suppressed replication of the plasmid in TC7 monkey cells, although the plasmids expressed replication-competent T antigen. Rat cells were transformed with linearized wild-type SV40 plasmid DNA with or without fragments of the mouse DNA in cis. Although all of the rat cell lines expressed approximately equal amounts of T antigen and p53, transformants carrying SV40 DNA linked to either of the same two replication suppressor fragments produced significantly less free SV40 DNA after fusion with permissive cells than those transformed by SV40 DNA without a cellular insert or with a cellular insert lacking suppressor activity. The results suggest that two independent segments of cellular DNA act in cis to suppress SV40 replication in vivo, either as a plasmid or integrated in chromosomal DNA.     

Transgene Coplacement and High Efficiency Site-Specific Recombination with the Cre/Loxp System in Drosophila

Studies of gene function and regulation in transgenic Drosophila are often compromised by the possibility of genomic position effects on gene expression. We have developed a method, called transgene coplacement, in which any two sequences can be positioned at exactly the same site and orientation in the genome. Transgene coplacement makes use of the bacteriophage P1 system of Cre/loxP site-specific recombination, which we have introduced into Drosophila. In the presence of a cre transgene driven by a dual hsp70-Mos1 promoter, a white reporter gene flanked by loxP sites is excised with virtually 100% efficiency both in somatic cells and in germ cells. A strong maternal effect, resulting from Cre recombinase present in the oocyte, is observed as white or mosaic eye color in F(1) progeny. Excision in germ cells of the F(1) yields a strong grand-maternal effect, observed as a highly skewed ratio of eye-color phenotypes in the F(2) generation. The excision reactions of Cre/loxP and the related FLP/FRT system are used to create Drosophila lines in which transgenes are at exactly allelic sites in homologous chromosomes.     

Phylogeny and physiology of Drosophila opsins

Phylogenetic and physiological methods were used to study the evolution of the opsin gene family in Drosophila. A phylogeny based on DNA sequences from 13 opsin genes including representatives from the two major subgenera of Drosophila shows six major, well-supported clades: The blue opsin clade includes all of the Rhl and Rh2 genes and is separated into two distinct subclades of Rhl sequences and Rh2 sequences; the ultraviolet opsin clade includes all Rh3 and Rh4 genes and bifurcates into separate Rh3 and Rh4 clades. The duplications that generated this gene family most likely took place before the evolution of the subgenera Drosophila and Sophophora and their component species groups. Numerous changes have occurred in these genes since the duplications, including the loss and/or gain of introns in the different genes and even within the Rhl and Rh4 clades. Despite these changes, the spectral sensitivity of each of the opsins has remained remarkably fixed in a sample of four species representing two species groups in each of the two subgenera. All of the strains that were investigated had R1-6 (Rhl) spectral sensitivity curves that peaked at or near 480 nm, R7 (Rh3 and Rh4) peaks in the ultraviolet range, and ocellar (Rh2) peaks near 420 nm. Each of the four gene clades on the phylogeny exhibits very conservative patterns of amino acid replacement in domains of the protein thought to influence spectral sen sitivity, reflecting strong constraints on the spectrum of light visible to Drosophila.     

Significant hydrogen exchange protection in GroEL-bound DHFR is maintained during iterative rounds of substrate cycling.

An unresolved key issue in the mechanism of protein folding assisted by the molecular chaperone GroEL is the nature of the substrate protein bound to the chaperonin at different stages of its reaction cycle. Here we describe the conformational properties of human dihydrofolate reductase (DHFR) bound to GroEL at different stages of its ATP-driven folding reaction, determined by hydrogen exchange labeling and electrospray ionization mass spectrometry. Considerable protection involving about 20 hydrogens is observed in DHFR bound to GroEL in the absence of ATP. Analysis of the line width of peaks in the mass spectra, together with fluorescence quenching and ANS binding studies, suggest that the bound DHFR is partially folded, but contains stable structure in a small region of the polypeptide chain. DHFR rebound to GroEL 3 min after initiating its folding by the addition of MgATP was also examined by hydrogen exchange, fluorescence quenching, and ANS binding. The results indicate that the extent of protection of the substrate protein rebound to GroEL is indistinguishable from that of the initial bound state. Despite this, small differences in the quenching coefficient and ANS binding properties are observed in the rebound state. On the basis of these results, we suggest that GroEL-assisted folding of DHFR occurs by minor structural adjustments to the partially folded substrate protein during iterative cycling, rather than by complete unfolding of this protein substrate on the chaperonin surface.