Biomedical subjects

G C Shore

Publications and source records attributed to G C Shore.

At least 55 records · Page 3Linked to original sources

PubMed1993

Targeting of Bcl-2 to the mitochondrial outer membrane by a COOH-terminal signal anchor sequence.

The protooncogene product Bcl-2 is an integral membrane protein that functions as a suppressor of programmed cell death. It contains a single predicted transmembrane segment located at its COOH terminus. Here, we show that the transmembrane domain of human Bcl-2 functions as a mitochondrial signal anchor sequence that targets and inserts the protein into the outer membrane in an Ncyto-C(in) orientation, leaving the bulk of the polypeptide facing the cytosol. Deletion of the COOH-terminal 22 amino acids of Bcl-2 abrogated protein targeting, whereas fusion of this domain to the COOH terminus of dihydrofolate reductase resulted in targeting and insertion of the hybrid protein into the outer membrane in a manner similar to that of Bcl-2. The sequence of the hydrophobic core of the Bcl-2 signal anchor is similar to the corresponding region of the NH2-terminal signal anchor of the mitochondrial outer membrane protein in yeast, Mas70p. A synthetic peptide comprising the Mas70p signal anchor sequence effectively competed for insertion of Bcl-2 into the outer membrane but had no effect on the comparatively low association that Bcl-2 makes with endoplasmic reticulum microsomes. Insertion of Bcl-2 into the mitochondrial outer membrane is mechanistically different than its association with microsomes.

Amino Acid Sequence↗

PubMed1993

The signal anchor sequence of mitochondrial Mas70p contains an oligomerization domain.

pOMD29 is a mitochondrial precursor protein that contains the NH2-terminal signal anchor sequence of Mas70p fused to dihydrofolate reductase. The signal anchor mediates insertion of pOMD29 into the outer mitochondrial membrane in the Nin-Ccyto orientation. Following import in vitro, pOMD29 was chemically cross-linked, via a unique cysteine residue adjacent to the signal anchor (residue 34), to form a product that was approximately twice the size of pOMD29. The cross-linked product was a dimer of pOMD29, as judged by the following. 1) It exhibited the same charge:mass ratio as pOMD29. 2) Formation of radioactive cross-linked product containing 35S-labeled pOMD29 was stimulated by co-import with unlabeled pOMD29. 3) Co-import of pOMD29 with a modified pOMD29 that contains two copies of dihydrofolate reductase resulted in formation of the predicted homo- and heterodimers. Cross-linking of pOMD29 was unaffected by concentrations of methotrexate that lock the dihydrofolate reductase moiety into its native monomeric conformation, indicating that oligomerization was mediated by the signal anchor rather than by the cytosolic domain of pOMD29. The predicted transmembrane core of the signal anchor sequence contains structural motifs similar to those found in a variety of signal-transducing cell surface receptors that dimerize through their transmembrane segments.

Base Sequence↗

PubMed1992

The carbamyl phosphate synthetase promoter contains multiple binding sites for C/EBP-related proteins.

The promoter of the gene (CPS) encoding rat carbamyl phosphate synthetase I has been mapped 5' to a segment of about 525 nucleotides upstream from the transcription start point and, when analyzed in liver nuclear extracts, contained six well-defined protein-recognition elements, designated CPS sites I-VI. All six elements were recognized, with varying affinities, by CAAT and enhancer-binding protein (C/EBP alpha) produced in bacteria. Oligodeoxyribonucleotides corresponding to CPS site II or to the C/EBP alpha-recognition element of the ALB promoter, site D, competed with the six CPS-promoter elements in footprinting assays. However, mutagenesis of the C/EBP alpha-recognition element, 5'-GTTGCAAC, at the core of site II was sufficient to abolish transactivation of the CPS promoter by C/EBP alpha in co-transfected HepG2 cells. These findings indicate that the CPS promoter contains multiple recognition elements for factors with DNA-binding specificities similar to C/EBP proteins. Activation by C/EBP alpha, however, requires promoter site II.

Animals↗

PubMed1992

Factors interacting with the rat carbamyl phosphate synthetase promoter in expressing and nonexpressing tissues.

The proximal promoter of the rat carbamyl phosphate synthetase-encoding gene (CPS) contains at least six potential cis-acting regulatory elements (sites I-VI), as judged by DNase I footprint analysis using rat liver nuclear extracts; all six regions bind proteins with DNA recognition properties similar to those of CAAT and enhancer-binding protein alpha (C/EBP alpha) [Lagacé et al., Gene 118 (1992) 231-238]. In contrast, nuclear extracts from kidney, brain and spleen contain proteins that recognize CPS promoter sites II, V and VI, but not sites I, III and IV. Mutation of the octameric sequence (5'-GTTGCAAC) within site II, which is a recognition element for C/EBP alpha, abolished binding of nuclear proteins to site II oligodeoxyribonucleotides (oligos) in all tissues. As well, the site II mutation reduced the level of in vitro transcription from the CPS promoter by about 50% in liver and spleen nuclear extracts, but had a negligible effect in brain and kidney extracts. The fact that promoter activity was observed in extracts of tissues that do not express the endogenous CPS gene (i.e., brain, kidney and spleen) indicates that these tissues, nevertheless, contain factors with the potential to activate transcription through a limited number of CPS promoter elements. Tissue-specific regulation, therefore, must involve steps to prevent these factors from acting on the endogenous CPS promoter in situ.

Animals↗

PubMed1992

Reversal of the orientation of an integral protein of the mitochondrial outer membrane.

The NH2-terminus of the signal-anchor sequence of an integral, bitopic protein of the outer mitochondrial membrane was extended both in amino acid length (from 11 to 38 amino acids) and net charge (from +4 to +8)--changes that confer on the NH2-terminus characteristics of a strong matrix-targeting signal. The protein was inserted into the outer membrane but in an inverted orientation (Ncyto-Cin). These findings suggest that, in common with other membrane systems, the orientation of a protein in the outer mitochondrial membrane can be determined by a signal that causes retention of the NH2-terminus on the cytosolic side of the membrane.

Amino Acid Sequence↗

PubMed1992

Protein sorting between mitochondrial outer and inner membranes. Insertion of an outer membrane protein into the inner membrane.

The amino terminal 29 amino acids of the outer mitochondrial membrane protein of yeast, OMM70 (MAS70), consisting of the targeting and membrane anchor domains, has been fused to a reporter protein, dihydrofolate reductase. The hybrid protein, designated pOMD29, was efficiently imported into the outer membrane of rat heart mitochondria by a process dependent on ATP and proteinase-sensitive components on the surface of the organelle, and in which the orientation of the native protein was retained. To determine if the protein translocation machinery of the inner membrane is also capable of recognizing and inserting pOMD29, direct access to the intermembrane space was provided to pOMD29 by selectively rupturing the mitochondrial outer membrane by osmotic shock. In this system, the outer membrane binding site for matrix-destined precursor proteins can be bypassed, and efficient import restored to proteinase-pretreated mitochondria. pOMD29 was imported into the inner membrane of osmotically-shocked mitochondria, mediated by protein components. The outer membrane orientation of pOMD29 was conserved when inserted into the inner membrane but, unlike the outer membrane, import into the inner membrane required delta psi. We conclude that the protein translocation machinery of the mitochondrial inner membrane is capable of recognizing and inserting a protein whose topogenic information otherwise results in insertion of the protein to the outer membrane. The significance of these findings for sorting of proteins between the mitochondrial inner and outer membranes is discussed.

Amino Acid Sequence↗

PubMed1992

A signal-anchor sequence selective for the mitochondrial outer membrane.

pOMD29 is a hybrid protein containing the NH2-terminal topogenic sequence of a bitopic, integral protein of the outer mitochondrial membrane in yeast, OMM70, fused to dihydrofolate reductase. The topogenic sequence consists of two structural domains: an NH2-terminal basic region (amino acids 1-10) and an apolar region which is the predicted transmembrane segment (amino acids 11-29). The transmembrane segment alone was capable of targeting and inserting the hybrid protein into the outer membrane of intact mitochondria from rat heart in vitro. The presence of amino acids 1-10 enhanced the rate of import, and this increased rate depended, in part, on the basic amino acids located at positions 2, 7, and 9. Deletion of a large portion of the transmembrane segment (amino acids 16-29) resulted in a protein that exhibited negligible import in vitro. Insertion of pOMD29 into the outer membrane was not competed by import of excess precursor protein destined for the mitochondrial matrix, indicating that the two proteins may have different rate-limiting steps during import. We propose that the structural domains within amino acids 1-29 of pOMD29 cooperate to form a signal-anchor sequence, the characteristics of which suggest a model for proper sorting to the mitochondrial outer membrane.

Amino Acid Sequence↗

PubMed1990

Mitochondrial precursor protein. Effects of 70-kilodalton heat shock protein on polypeptide folding, aggregation, and import competence.

A hybrid precursor protein constructed by fusing the mitochondrial matrix-targeting signal of rat preornithine carbamyl transferase to murine cytosolic dihydrofolate reductase (designated pO-DHFR) was expressed in Escherichia coli. Following purification under denaturing conditions, pO-DHFR was capable of membrane translocation when diluted directly into import medium containing purified mitochondria but lacking cytosolic extracts. This import competence was lost with time, however, when the precursor was diluted and preincubated in medium lacking mitochondria, unless cytosolic proteins (provided by rabbit reticulocyte lysate) were present. Identical results were obtained for purified precursor made by in vitro translation. The ability of the cytosolic proteins to maintain the purified precursor in an import-competent state was sensitive to protease, N-ethylmaleimide (NEM), and was heat labile. Further, this activity appeared to be signal sequence dependent. ATP was not required for the maintenance of pO-DHFR competence, nor did purified 70-kDa heat shock protein (the constitutive form of Hsp70) substitute for this activity. Interestingly, however, purified Hsp70 prevented aggregation of the precursor in an ATP-dependent manner and, as well, retarded the apparent rate and extent of pO-DHFR folding. Partial purification of reticulocyte lysate proteins indicated that competence activity resides within a large mass protein fraction (200-250 kDa) that contains Hsp70. Sucrose density gradient analysis revealed that pO-DHFR reversibly interacts with components of this fraction. Pretreatment of the fraction with NEM, however, significantly stabilized the subsequent formation of a complex with the precursor. The results indicate that Hsp70 can retard precursor polypeptide folding and prevent precursor aggregation; however, by itself, Hsp70 cannot confer import competence to pO-DHFR. Maintenance of import competence correlates with interactions between the precursor and an NEM-sensitive cytosolic protein fraction. Efficient dissociation of the precursor from this complex appears to require a reactive thiol moiety on the cytosolic protein(s).

Adenosine Triphosphate↗

PubMed1990

Import of precursor proteins into mitochondria: site of polypeptide unfolding.

The matrix-targeting signal of mitochondrial preornithine carbamyl transferase has been fused to either murine dihydrofolate reductase (pODHFR) or bacterial chloramphenicol acetyltransferase (pOCAT). Loosening of the tightly folded "native" structure of the two proteins following their synthesis in a rabbit reticulocyte lysate was assayed by the acquisition of protease sensitivity (pODHFR and pOCAT) or by the loss of enzyme activity (pOCAT). By these criteria, the bulk population of both precursor proteins was tightly folded following release from the ribosome, even in the presence of ATP and excess reticulocyte lysate. Neither protein unfolded as a consequence of binding to the surfaces of anionic liposomes or intact mitochondria. However, a non-native form of full-length pOCAT, exhibiting a loss of enzymatic activity and an enhanced protease sensitivity, was detected in association with a submitochondrial fraction that banded between the inner and outer mitochondrial membrane fractions on sucrose density gradients. Delivery of the precursor molecule to this position required ATP and a proteinaceous component on the surface of the organelle.

Animals↗

PubMed1990

An amino-terminal signal sequence abrogates the intrinsic membrane-targeting information of mitochondrial uncoupling protein.

Mitochondrial uncoupling protein, a polytopic integral protein of the inner membrane, is initially made in the cytoplasm as a soluble polypeptide (307 amino acids) lacking a cleavable targeting (signal) peptide. Earlier studies (Liu, X., Bell, A. W., Freeman, K. B., and Shore, G. C. (1988) J. Cell Biol. 107, 503-509) identified internal regions of the molecule that are critical for targeting and membrane insertion. Here, we demonstrate that the ability of uncoupling protein to insert into the inner membrane is abrogated when the molecule is fused behind the matrix-targeting signal of preornithine carbamyltransferase; the hybrid protein was imported across the inner membrane and deposited in the matrix where it was processed. In this context, however, the processed product remained in the matrix and was incapable of inserting into the inner membrane.

Animals↗

PubMed1989

Import of a mutant mitochondrial precursor fails to respond to stimulation by a cytosolic factor.

Import of the precursor to ornithine carbamyltransferase is stimulated by a partially-purified, NEM-sensitive soluble factor from rabbit reticulocyte lysate. A mutant in which the carboxy-terminal 73 amino acids were deleted, had a sharply reduced response to this factor. The NEM-sensitive, import-stimulating factor interacts with the surface of mitochondria in the absence of precursor protein. Thus reticulocyte lysate contains an NEM-sensitive, import stimulating factor which interacts both with the surface of mitochondria and whose activity appears to be dependent upon the structure of the mature portion of the precursor.

Amino Acid Sequence↗

PubMed1989

Activity of the carbamyl phosphate synthetase I promoter in liver nuclear extracts is dependent on a cis-acting C/EBP recognition element.

We have identified an essential cis element in the proximal promoter region of the rat carbamyl phosphate synthetase I (CPSI) gene that is requisite for promoter activity in liver nuclear extracts. Excess synthetic oligonucleotides specifying this region abolished promoter-dependent in vitro transcription. We show that C/EBP, a nuclear factor enriched in liver but found as well in other tissues, such as gut, fat, and lung, interacts with an inverted repeat, GTTGCAAC, at the core of the essential cis element. In brain, a tissue that did not express CPSI or contain significant levels of C/EBP, a different factor was capable of binding at or near the C/EBP recognition element. Activity of the CPSI promoter in liver nuclear extracts was also dependent on sequences 5' to the C/EBP motif; presumably, factors binding to elements within this upstream region are instrumental in restricting CPSI gene expression to liver and intestinal mucosa.

Animals↗

PubMed1988

Identification of hydrophobic residues in the signal sequence of mitochondrial preornithine carbamyltransferase that enhance the rate of precursor import.

Previous studies employing circular dichroism and resonance energy transfer techniques have demonstrated that the signal peptide of mitochondrial preornithine carbamyltransferase (pOCT) has the potential to interact with the surface of an anionic phospholipid membrane via a short amphiphilic helical domain. Here we have used predictive secondary structure computations as a guide to localize the putative membrane binding region in the pOCT signal sequence and demonstrate that replacement of leucine residues at positions 5, 8, and 9 with the less hydrophobic residue, alanine, significantly reduces the rate of precursor import (4-5-fold compared to wild type); the amino acid substitutions had little effect, however, on the ability of a mitochondrial matrix extract to process the mutant precursor polypeptide. The mutant precursor bound to anionic liposomes with a lower affinity compared to wild-type pOCT and was inhibited to a lesser extent than pOCT during import into mitochondria in the presence of varying concentrations of liposomes. Taken together, the results suggest that this small region of the pOCT signal sequence, containing a limited number of critical hydrophobic residues, contributes to the optimal rate of precursor import, perhaps by functioning as a membrane surface-seeking entity.

Amino Acid Sequence↗

PubMed1988

Protein sorting between mitochondrial membranes specified by position of the stop-transfer domain.

Recently, we fused a matrix-targeting signal to a large fragment of vesicular stomatitis virus G protein, which contains near its COOH-terminus a well-characterized endoplasmic reticulum (ER) stop-transfer sequence; the hybrid G protein was sorted to the inner mitochondrial membrane (Nguyen, M., and G. C. Shore. 1987. J. Biol. Chem. 262:3929-3931). Here, we show that the 19 amino acid G stop-transfer domain functions in an identical fashion when inserted toward the COOH-terminus of an otherwise normal matrix precursor protein, pre-ornithine carbamyl transferase; after import, the mutant protein was found anchored in the inner membrane via the stop-transfer sequence, with its NH2 terminus facing the matrix and its short COOH-terminal tail located in the intermembrane space. However, when the G stop-transfer sequence was placed near the NH2 terminus, the protein was inserted into the outer membrane, in the reverse orientation (NH2 terminus facing out, with a large COOH-terminal fragment located in the intermembrane space). These observations for mitochondrial topogenesis can be explained by a simple extension of existing models for ER sorting.

Amino Acid Sequence↗

PubMed1988

Topogenesis of mitochondrial inner membrane uncoupling protein. Rerouting transmembrane segments to the soluble matrix compartment.

Brown adipose tissue uncoupling protein (UCP), an integral polytopic protein of the mitochondrial inner membrane, is composed of at least six transmembrane segments whose net hydrophobic character derives from paired amphiphilic helices. The protein is synthesized in the cytoplasm as a polypeptide (307 amino acids) lacking a cleavable targeting (signal) peptide. Deletion mutagenesis and fusion protein constructions revealed the existence of at least two import signals: one lying between UCP precursor amino acids 13-105 and the other downstream of position 101. The former resulted in both targeting and membrane insertion of a fusion protein, whereas the latter targeted UCP 102-307 into the organelle but failed to result in membrane insertion. When a strong matrix-targeting signal derived from precarbamoyl phosphate synthetase was fused to UCP amino acids 169-307 or 52-307 (containing three and five transmembrane domains, respectively), the fusion proteins were efficiently imported to the soluble matrix compartment where correct signal cleavage took place. We suggest that assembly of UCP into the inner membrane follows a coordinate insertion pathway for integration and may use more than one signal sequence to achieve this. In this respect, it might share certain mechanistic features with the insertion of polytopic proteins into the endoplasmic reticulum. The data also suggest, however, that integration of the amino-terminal third of UCP into the inner membrane may be required to help or enhance insertion of the remaining UCP transmembrane domains.

Adipose Tissue, Brown↗

PubMed1987

Rat carbamyl-phosphate synthetase I gene. Promoter sequence and tissue-specific transcriptional regulation in vitro.

The region flanking the 5'-end of the rat gene encoding the cytoplasmic precursor of carbamyl-phosphate synthetase I, a mitochondrial matrix enzyme, has been cloned and partially characterized. S1 nuclease and primer extension analyses position the starts of transcription 138-140 nucleotides upstream of the translation initiation codon. Exon 1 contains this untranslated sequence and extends downstream to include the coding region for the pre-enzyme signal peptide (38 amino acids) plus 4 amino acids from the amino terminus of the mature protein. The 5'-flanking sequence contains typical promoter elements, including putative TATA and CAAT motifs at -21 and -82 nucleotides, respectively. In addition, several copies of consensus sequences corresponding to the H4TF-1 recognition element, GATTTC, together with the enhancer-like octamer, ATTTGCAT, are also present. Carbamyl-phosphate synthetase I is a cell-type specific enzyme, being expressed only in hepatocytes and epithelial cells of the intestinal mucosa. It is also synthesized at relatively high levels in the hepatoma cell line, Hep G2. Employing pCPS2.1, a minigene containing the promoter and part of exon 1, we show that nuclear extracts from Hep G2 support accurate carbamyl-phosphate synthetase I gene transcription in vitro. No such activity was observed, however, in extracts from HeLa, a cell line which does not express carbamyl-phosphate synthetase I.

Animals↗

PubMed1987

Import of hybrid vesicular stomatitis G protein to the mitochondrial inner membrane.

cDNA fusions were employed to construct a 35-kDa hybrid protein bearing the amino-terminal signal sequence of pre-ornithine carbamoyltransferase (pOCT), a mitochondrial matrix enzyme, fused to the carboxyl-terminal half of vesicular stomatitis virus G protein (VSV G). Following transcription-translation in vitro, the hybrid protein was imported by purified mitochondria and processed at its amino terminus by the matrix processing enzyme. The protein, however, remained anchored in the mitochondrial inner membrane, apparently in a transmembrane configuration, via the hydrophobic VSV G stop-transfer domain; a small portion (approximately 2 kDa) of the G protein fragment was accessible to protease digestion only after selective permeabilization of the mitochondrial outer membrane with digitonin, a finding consistent with localization of the extreme carboxyl-terminal cytoplasmic tail of G in the intermembrane space. The results demonstrate that the membrane-anchor domain of VSV G can function in a post-translational manner and can operate in membranes other than those derived from the endoplasmic reticulum. However, it appears to be selectively recognized as a stop-transfer signal by the translocation machinery of the mitochondrial inner, rather than outer, membrane.

Animals↗

PubMed1987

The interaction of a synthetic mitochondrial signal peptide with lipid membranes is independent of transbilayer potential.

We have used fluorescence measurements and assays of vesicle disruption (contents leakage) to monitor the interaction between lipid vesicles and a synthetic peptide corresponding to the N-terminal 27 amino acids of rat mitochondrial pre-ornithine carbamyltransferase (pOCT). This peptide and two fluorescent derivatives bind reversibly to vesicles composed of neutral and anionic phospholipids with increasing affinity as the proportion of anionic lipids in the vesicles increases. The affinity of the peptide for lipid vesicles is unaffected by the presence of a transbilayer potential (inside negative) of at least -80 mV across the vesicle membranes. Our results support the proposal that the signal sequence of pOCT may promote an initial association of the precursor protein with mitochondrial membranes prior to binding to a specific receptor. However, we find no evidence that the pOCT signal sequence can subsequently undergo transfer into or across the lipid bilayer, even in the presence of a transmembrane potential of the magnitude previously found to support the import of precursor proteins into mitochondria.

Animals↗