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M G Douglas

Publications and source records attributed to M G Douglas.

At least 19 recordsLinked to original sources
PubMed2001

Biogenesis of porin of the outer mitochondrial membrane involves an import pathway via receptors and the general import pore of the TOM complex.

Porin, also termed the voltage-dependent anion channel, is the most abundant protein of the mitochondrial outer membrane. The process of import and assembly of the protein is known to be dependent on the surface receptor Tom20, but the requirement for other mitochondrial proteins remains controversial. We have used mitochondria from Neurospora crassa and Saccharomyces cerevisiae to analyze the import pathway of porin. Import of porin into isolated mitochondria in which the outer membrane has been opened is inhibited despite similar levels of Tom20 as in intact mitochondria. A matrix-destined precursor and the porin precursor compete for the same translocation sites in both normal mitochondria and mitochondria whose surface receptors have been removed, suggesting that both precursors utilize the general import pore. Using an assay established to monitor the assembly of in vitro-imported porin into preexisting porin complexes we have shown that besides Tom20, the biogenesis of porin depends on the central receptor Tom22, as well as Tom5 and Tom7 of the general import pore complex (translocase of the outer mitochondrial membrane [TOM] core complex). The characterization of two new mutant alleles of the essential pore protein Tom40 demonstrates that the import of porin also requires a functional Tom40. Moreover, the porin precursor can be cross-linked to Tom20, Tom22, and Tom40 on its import pathway. We conclude that import of porin does not proceed through the action of Tom20 alone, but requires an intact outer membrane and involves at least four more subunits of the TOM machinery, including the general import pore.

Amino Acid Sequence↗
PubMed2000

ASC1/RAS2 suppresses the growth defect on glycerol caused by the atp1-2 mutation in the yeast Saccharomyces cerevisiae.

To better define the regulatory role of the F(1)-ATPase alpha-subunit in the catalytic cycle of the ATP synthase complex, we isolated suppressors of mutations occurring in ATP1, the gene for the alpha-subunit in Saccharomyces cerevisiae. First, two atp1 mutations (atp1-1 and atp1-2) were characterized that prevent the growth of yeast on non-fermentable carbon sources. Both mutants contained full-length F(1)alpha-subunit proteins in mitochondria, but in lower amounts than that in the parental strain. Both mutants exhibited barely measurable F(1)-ATPase activity. The primary mutations in atp1-1 and atp1-2 were identified as Thr(383) --> Ile and Gly(291) --> Asp, respectively. From recent structural data, position 383 lies within the catalytic site. Position 291 is located near the region affecting subunit-subunit interaction with the F(1)beta-subunit. An unlinked suppressor gene, ASC1 (alpha-subunit complementing) of the atp1-2 mutation (Gly(291) --> Asp) restored the growth defect phenotype on glycerol, but did not suppress either atp1-1 or the deletion mutant Deltaatp1. Sequence analysis revealed that ASC1 was allelic with RAS2, a G-protein growth regulator. The introduction of ASC1/RAS2 into the atp1-2 mutant increased the F(1)-ATPase enzyme activity in this mutant when the transformant was grown on glycerol. The possible mechanisms of ASC1/RAS2 suppression of atp1-2 are discussed; we suggest that RAS2 is part of the regulatory circuit involved in the control of F(1)-ATPase subunit levels in mitochondria.

Adaptor Proteins, Signal Transducing↗
PubMed2000

Molecular interactions of cancer and age.

The authors believe that the aging process--the loss of youthful resilience--is caused by the decline of many hormones. By restoring these hormone levels, the decay associated with old age can be eliminated and in some cases, perhaps reversed. The hormones that naturally occur in the body should be replenished through a medically sound regimen. The hormones must work together. The data indicate that aging occurs at two levels: systemically and cellularly. Systemic controls regulate the rates of intracellular enzymatic processes that accelerate to protect against aging.

Adult↗
PubMed1996

Specific targeting of ISP6 to mitochondria is mediated by sequences other than its amino terminus.

Most proteins synthesized in cytoplasm target to mitochondria through sequences at their amino termini. However, a previous study suggests that the native carboxyl terminus of ISP6 might be critical of its specific delivery. Here we investigated the sequence directing ISP6 to yeast mitochondrial outer membrane. Unlike mitochondrial presequences, a region at the amino terminus of ISP6 is dispensable for importing the rest of the protein. The carboxyl-terminal end and the nearby transmembrane region of ISP6 are essential to direct the protein exclusively to its correct membrane destination. ISP6 thus may be directed to mitochondria by an unusual sequence.

Amino Acid Sequence↗
PubMed1996

A conserved HPD sequence of the J-domain is necessary for YDJ1 stimulation of Hsp70 ATPase activity at a site distinct from substrate binding.

The 46-kDa protein YDJ1 is one of several known yeast homologues of the Escherichia coli DnaJ protein. Like all J homologues, it shares homology with the highly conserved NH2-terminal "J-domain" of DnaJ. A component of the DnaK (Hsp70) chaperone machinery that mediates protein folding, DnaJ is necessary for survival at elevated temperatures. It stimulates ATP hydrolysis by DnaK and effects the release of DnaK-bound polypeptides. Previous genetic and biochemical studies indicate that the J-domain is necessary for these functions. Using peptides corresponding to J-domain sequence, we show that a peptide containing the highly conserved His-Pro-Asp sequence at positions 34-36 in the J-domain competes off YDJ1 stimulation of Hsp70 ATPase activity. Inhibitory concentrations of peptide do not prevent binding of folding substrates, therefore YDJ1 must interact with Hsp70 at a site distinct from that for substrate binding. This interaction is critical for Hsp70 activity, since a mutant YDJ1 protein harboring a H34Q change (ydj1Q34) stimulates neither Hsp70 ATPase nor substrate release. The importance of the proper function of this region of the protein is supported by the poor growth and temperature-sensitive phenotype of yeast expressing ydj1Q34.

Adenosine Triphosphatases↗
PubMed1996

Determinants of success of color-flow duplex-guided compression repair of femoral pseudoaneurysms.

BACKGROUND: Ultrasonography-guided compression repair is reported to be effective therapy for femoral pseudoaneurysms that develop after catheterization procedures. This study summarizes our experience with color-flow duplex-guided repair of these lesions. METHODS: A retrospective chart review of all patients who underwent this procedure was undertaken, with statistical analysis to identify factors associated with success. RESULTS: Compression repair of 69 pseudoaneurysms was attempted. Pseudoaneurysms developed after therapeutic catheterization in 48 patients and after diagnostic procedures in 21. Sites of arterial puncture were the common femoral artery in 59 patients and the superficial femoral or profunda femoris arteries in 10. Diameters of the pseudoaneurysms ranged from 3 to 60 mm (mean, 28 mm). Compression was attempted at a mean of 5 days (range, 1 to 21 days) after catheterization. Compression produced complete thrombosis of the pseudoaneurysm at the initial attempt in 43 (62%) of 69 patients. With repeated attempts the ultimate success was 47 (68%) of 69. Success was achieved in 44 (75%) of 59 common femoral pseudoaneurysms but in only 3 (30%) of 10 superficial femoral or profunda femoris lesions (p = 0.009). Anticoagulation, sheath size, pseudoaneurysm chamber size, and time between catheterization and compression were not significantly different between lesions that were successfully compressed and those that were not. No ischemic or embolic complications were observed. CONCLUSIONS: Color-flow duplex-guided compression repair can be safely attempted as the initial therapy for all uncomplicated pseudoaneurysms arising from the common femoral artery after catheterization, with the expectation of success in most.

Adult↗
PubMed1995

Biogenesis of ISP6, a small carboxyl-terminal anchored protein of the receptor complex of the mitochondrial outer membrane.

To study the biogenesis of ISP6, an outer membrane component of the mitochondrial protein translocation complex, two fusion proteins have been made by fusing ISP6 to either the carboxyl- or amino-terminal end of the mouse dihydrofolate reductase (DHFR). In vitro import experiments showed that when DHFR was placed at the carboxyl-terminal end of ISP6, the resulting fusion protein 6-DHFR inserted into mitochondrial membrane less efficiently than the other form of the fusion proteins. In vivo this fusion protein lost its ability to suppress the temperature-sensitive phenotype of an isp42 mutant, while the other fusion protein DHFR-6, which was found targeted correctly to mitochondria, suppressed the mutant as well as the wild-type ISP6. Further analysis showed that the binding and insertion of DHFR-6 to mitochondrial outer membrane was not affected by deletion of either of the two mitochondrial protein receptors or by the predigestion of mitochondrial surface proteins prior to import. Additional data indicated that ISP42, which closely associates with ISP6 in the translocation complex, does not likely play the role of a targeting partner for ISP6. In summary, these data suggest that ISP6 may target to mitochondria by sequences at its carboxyl terminus and that the import process of ISP6 is most likely distinct from that of most other mitochondrial precursors, which are recognized by protein receptors on mitochondrial surface.

Amino Acid Sequence↗
PubMed1995

An argument against routine percutaneous biopsy, ERCP, or biliary stent placement in patients with clinically resectable periampullary masses: a surgical perspective.

Improve resolution of computed tomography (CT) and ultrasonography allows us to visualize the proximal extent of biliary obstruction and the presence of a periampullary mass in most patients with malignant extrahepatic biliary obstruction. Our purpose in this report is to challenge the need for preoperative percutaneous biopsy, endoscopic retrograde cholangiopancreatography, or preoperative placement of a biliary endoprosthesis in the good-risk patient in whom the imaging procedure clearly defines a periampullary mass and the proximal extent (hepatic extent) of biliary obstruction. We recently managed three patients in whom one of these invasive procedures led to a complication that delayed, prevented, or complicated appropriate operative resection of a pancreatic neoplasm. Because a negative percutaneous biopsy, cholangiographic imaging of a dilated bile/pancreatic duct clearly seen on CT or ultrasonography, or short-term preoperative biliary decompression does not alter the decision for operative exploration and may cause complications, we argue against their use in the good-risk patient with both extrahepatic biliary obstruction and a periampullary pancreatic mass well delineated on noninvasive imaging.

Aged↗
PubMed1995

RPM2, independently of its mitochondrial RNase P function, suppresses an ISP42 mutant defective in mitochondrial import and is essential for normal growth.

RPM2 is identified here as a high-copy suppressor of isp42-3, a temperature-sensitive mutant allele of the mitochondrial protein import channel component, Isp42p. RPM2 already has an established role as a protein component of yeast mitochondrial RNase P, a ribonucleoprotein enzyme required for the 5' processing of mitochondrial precursor tRNAs. A relationship between mitochondrial tRNA processing and protein import is not readily apparent, and, indeed, the two functions can be separated. Truncation mutants lacking detectable RNase P activity still suppress the isp42-3 growth defect. Moreover, RPM2 is required for normal fermentative yeast growth, even though mitochondrial RNase P activity is not. The portion of RPM2 required for normal growth and suppression of isp42-3 is the same. We conclude that RPM2 is a multifunctional gene. We find Rpm2p to be a soluble protein of the mitochondrial matrix and discuss models to explain its suppression of isp42-3.

Amino Acid Sequence↗
PubMed1994

Differential regulation of Hsp70 subfamilies by the eukaryotic DnaJ homologue YDJ1.

In Saccharomyces cerevisiae Ydj1p, a DnaJ homolog, is localized to the cytosol with the Ssa and Ssb Hsp70 proteins. Ydj1p helps facilitate polypeptide translocation across mitochondrial and endoplasmic reticulum membranes (Caplan, A. J., Cyr, D. M., and Douglas, M. G. (1992) Cell 71, 1143-1155) and can directly interact with Ssa1p to regulate chaperone activity (Cyr, D. M., Lu, X., and Douglas, M. G. (1992) J. Biol. Chem. 267, 20927-20931). In this study, the role of Ydj1p in modulating ATP-dependent reactions catalyzed by Ssa and Ssb Hsp70 proteins has been examined using purified components and compared with that of other Hsp70 homologs BiP and DnaK. Ssa1p, Ssa2p, and Ssb1/2p all formed stable complexes with the mitochondrial presequence peptide, F1 beta(1-51). ATP alone had only modest effects on polypeptide complex formation with Ssa1p and Ssa2p, but prevented the majority of polypeptide binding to BiP and DnaK. ATP by itself also reduced polypeptide binding to Ssb1/2p to a level that was intermediate between that observed for the Ssa Hsp70 proteins tested and BiP and DnaK. ATP hydrolysis by Ssa1p, Ssa2p, and Ssb1/2p occurred at similar rates. Ydj1p was a potent modulator of the both the ATPase and polypeptide binding activities of Ssa1p and Ssa2p. In contrast, Ydj1p had little effect on the ATPase and polypeptide binding activity of Ssb1/2p. Therefore the chaperone-related activities of Ssa and Ssb Hsp70 proteins exhibit significant differences in sensitivity to ATP and YDJ1p. These data indicate that regulation of Hsp70 activity by DnaJ homologs can be specific. The specificity of interactions between Ydj1p and the Ssa and Ssb Hsp70 proteins observed could contribute in determining the functional specificity of these chaperones in the cytosol. In related experiments, F1 beta(1-51) was found to reduce the extent to which Ydj1p stimulated Ssa1p ATPase activity. This effect correlated with the formation of F1 beta(1-51).Ssa1p complexes. We propose that intramolecular communication between the polypeptide binding, ATPase and DnaJ regulatory domains on Ssa1p plays a role in the regulation of chaperone activity.

Adenosine Triphosphatases↗
PubMed1994

DnaJ-like proteins: molecular chaperones and specific regulators of Hsp70.

The folding of proteins and the assembly of protein complexes within subcompartments of the eukaryotic cell is catalysed by different members of the Hsp70 protein family. The chaperone function of Hsp70 proteins in these events is regulated by members of the DnaJ-like protein family, which occurs through direct interaction of different Hsp70 and DnaJ-like protein pairs that appear to be specifically adapted to each other. This review highlights the diversity of functions of DnaJ-like proteins by using specific examples of DnaJ-Hsp70 interactions with polypeptides in yeast protein-biogenesis pathways.

Escherichia coli↗
PubMed1993

Site-directed mutagenesis of the yeast mitochondrial ADP/ATP translocator. Six arginines and one lysine are essential.

The ADP/ATP translocator mediates adenine nucleotide exchange across the inner mitochondrial membrane. ADP/ATP exchange is essential when yeast are grown on a non-fermentable carbon source such as glycerol, but it is not required for growth on glucose. Failure to grow on glycerol is therefore a phenotypic indicator of protein function, and it has been used here to screen site-directed mutants to identify functionally important amino acids in the yeast adenine nucleotide translocator (AAC2). Single mutations of all four charged amino acids in the transmembrane segments of AAC2 (K38A, R96D, R96H, R96L, R96P, R204L, R294A) resulted in loss of function, as did mutations in the matrix arginine cluster (R252I, R253I, R254I). Seven other residues were mutated without affecting growth on glycerol (C73S, C244S, C271S, K179M, K182I, P247G, W235F). The non-functional mutants have been used to select intragenic suppressors to gain further insight into the structure of this membrane transport protein.

Amino Acid Sequence↗
PubMed1993

Function-based mapping of the yeast mitochondrial ADP/ATP translocator by selection for second site revertants.

Structure-function relationships in the yeast adenine nucleotide carrier (AAC2) were probed by genetic selection techniques in Saccharomyces cerevisiae. As described in the preceding paper, yeast require a functional AAC2 (or AAC1) to grow on a non-fermentable carbon source. Mutants of AAC2 that could not grow on glycerol were subjected to selection for spontaneous suppressors. Ile mutants in the 100% conserved matrix Arg triplet R252 to R254 proved amenable to this approach, yielding colonies on glycerol plates at modest frequency. All mutants analyzed were single point mutations within the AAC2 gene at a different site than the Arg cluster. R254I gave the largest number (11) of unique revertants, while R253I gave only four and R252I gave none, thus, there was a gradient of effect in mutations of the Arg cluster. Unexpectedly, 14 of the 15 revertant mutations affected 13 different amino acids in a narrow sector of the AAC2 topological map, near the cytosolic surface. These mutants are proposed to be in, or very near, the membrane on the opposite side from the matrix Arg cluster. Helical wheel projections of the transmembrane segments show, with one exception, that mutations and charged residues fall within half of each helix. These mutants appear either to line the throat of the membrane channel, or to be involved in helix contacts near the cytosolic face of the inner mitochondrial membrane. These suppressors place further limitations on the organization of the nucleotide channel. We present a model of the AAC2 nucleotide channel based on these results. The region defined by these suppressors is dynamically linked to the Arg cluster through the function of the AAC2 protein. Discrete structures defined by multiple revertant mutations will likely be a common feature of similar regain-of-function schemes, especially when applied to membrane transport proteins. We propose these functionally mapped regions of proteins be named morphological units or morphs for short.

Amino Acid Sequence↗
PubMed1993

Genetic and biochemical characterization of ISP6, a small mitochondrial outer membrane protein associated with the protein translocation complex.

To search genetically for additional components of the protein translocation apparatus of mitochondria, we have used low fidelity PCR mutagenesis to generate temperature-sensitive mutants in the outer membrane translocation pore component ISP42. A high copy number suppressor of temperature-sensitive isp42 has been isolated and sequenced. This novel gene, denoted ISP6, encodes a 61 amino acid integral membrane protein of the mitochondrial outer membrane, which is oriented with its amino-terminus facing the cytosol. Disruption of the ISP6 gene is without apparent effect in wild type yeast cells, but is lethal in temperature-sensitive isp42 mutants. Immunoprecipitation of the gene product, ISP42p, from mitochondria solubilized under mild conditions reveals a multi-protein complex containing ISP6p and ISP42p.

Amino Acid Sequence↗