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F U Hartl

Publications and source records attributed to F U Hartl.

124 records · Page 7Linked to original sources
PubMed1989

Biochemical effects and zonal heterogeneity of peroxisome proliferation induced by perfluorocarboxylic acids in rat liver.

Rats were treated for 5 to 14 days with perfluoroacetate, perfluorobutyrate and perfluorooctanoate. Alterations in hepatic morphology with special reference to the peroxisomal compartment were investigated by light and electron microscopy following cytochemical staining of catalase activity with the alkaline 3,3'-diaminobenzidine medium. All three compounds induced hepatomegaly and peroxisome proliferation. Perfluorobutyrate and perfluorooctanoate were found to be more active than perfluoroacetate. Perfluorooctanoate-induced peroxisome proliferation was more prevalent in centrilobular than in periportal hepatocytes. Peroxisomes in centrilobular liver cells frequently were of round shape, exhibited diameters of up to 1.5 microns and were predominantly located within smooth endoplasmic reticulum-glycogen areas. In periportal cells, however, clusters of polymorphous peroxisomes ranging from 250 to 1,100 nm in diameter were observed at the periphery of smooth endoplasmic reticulum-glycogen regions. Peroxisome proliferation was accompanied by a change of peroxisomal and mitochondrial enzyme activities, in particular an increase in peroxisomal palmitoyl-CoA oxidation. Significant alterations in the concentration of peroxisomal matrix and membrane polypeptides were also noted. Within the first 2 days, perfluorooctanoate treatment exerted a strong hypolipidemic activity and both compounds perfluorooctanoate and perfluorobutyrate raised the level of hepatic free acid-soluble CoA nearly 10-fold as compared with control livers. The results suggest perfluorinated carboxylic acids to be model substances suitable to correlate biochemical and morphological parameters with the zonal heterogeneity of the peroxisomal compartment in rat liver. Due to the manifold hepatic effects, contact of humans with perfluorinated carboxylic acids or their metabolic precursors may represent a severe health risk.

Animals↗
PubMed1989

Translocation arrest by reversible folding of a precursor protein imported into mitochondria. A means to quantitate translocation contact sites.

Passage of precursor proteins through translocation contact sites of mitochondria was investigated by studying the import of a fusion protein consisting of the NH2-terminal 167 amino acids of yeast cytochrome b2 precursor and the complete mouse dihydrofolate reductase. Isolated mitochondria of Neurospora crassa readily imported the fusion protein. In the presence of methotrexate import was halted and a stable intermediate spanning both mitochondrial membranes at translocation contact sites accumulated. The complete dihydrofolate reductase moiety in this intermediate was external to the outer membrane, and the 136 amino acid residues of the cytochrome b2 moiety remaining after cleavage by the matrix processing peptidase spanned both outer and inner membranes. Removal of methotrexate led to import of the intermediate retained at the contact site into the matrix. Thus unfolding at the surface of the outer mitochondrial membrane is a prerequisite for passage through translocation contact sites. The membrane-spanning intermediate was used to estimate the number of translocation sites. Saturation was reached at 70 pmol intermediate per milligram of mitochondrial protein. This amount of translocation intermediates was calculated to occupy approximately 1% of the total surface of the outer membrane. The morphometrically determined area of close contact between outer and inner membranes corresponded to approximately 7% of the total outer membrane surface. Accumulation of the intermediate inhibited the import of other precursor proteins suggesting that different precursor proteins are using common translocation contact sites. We conclude that the machinery for protein translocation into mitochondria is present at contact sites in limited number.

Amino Acid Sequence↗
PubMed1989

Import of proteins into the various submitochondrial compartments.

Import of proteins into mitochondria can be subdivided into several distinct steps. (1) Mitochondrial proteins are synthesized on free ribosomes and are released into cytosolic pools. Nucleoside triphosphates are required to keep precursors in a conformation competent for import. (2) Precursors are directed to mitochondria by specific targeting signals (in most cases contained in N-terminal presequences) and by binding to receptors on the surface of the outer membrane. (3) Precursors interact with a component in the outer membrane which is believed to facilitate membrane insertion ('general insertion protein'). (4) Outer membrane proteins are then directly routed to their final location. Proteins of all other submitochondrial compartments are directed into translocation contact sites between outer and inner membranes. Transfer into contact sites is dependent on the membrane potential (delta psi) across the inner membrane. (5) Presequences of precursors are cleaved in the matrix by the mitochondrial processing peptidase in cooperation with the processing enhancing protein. (6) Precursors of the intermembrane space or the outer surface of the inner membrane have to be re-translocated back across the inner membrane ('conservative sorting').

Animals↗
PubMed1988

Cyclosporin A-binding protein (cyclophilin) of Neurospora crassa. One gene codes for both the cytosolic and mitochondrial forms.

Cyclophilin (cyclosporin A-binding protein) has a dual localization in the mitochondria and in the cytosol of Neurospora crassa. The two forms are encoded by a single gene which is transcribed into mRNAs having different lengths and 5' termini (approximately 1 and 0.8 kilobases). The shorter mRNA specifies the cytosolic protein consisting of 179 amino acids. The longer mRNA is translated into a precursor polypeptide with an amino-terminal extension of 44 amino acids which is cleaved in two steps upon entry into the mitochondrial matrix. Neurospora cyclophilin shows about 60% sequence homology to human and bovine cyclophilins.

Amino Acid Sequence↗
PubMed1988

Import of proteins into mitochondria: a multi-step process.

Translocation of precursor proteins from the cytosol into mitochondria is a multi-step process. The generation of translocation intermediates, i.e. the reversible accumulation of precursors at distinct stages of their import pathway into mitochondria ('translocation arrest'), has allowed the experimental characterization of distinct functional steps of protein import. These steps include: ATP-dependent unfolding of precursors; specific recognition of precursors by distinct receptors on the mitochondrial surface; interaction of precursors; specific recognition of precursors by distinct receptors on the mitochondrial surface; interaction of precursors with a general insertion protein ('GIP') in the outer mitochondrial membrane; membrane-potential-dependent translocation into the inner membrane at contact sites between both membranes; proteolytic processing of precursors; and intramitochondrial sorting of precursors via the matrix space ('conservative sorting'). The functional characteristics unveiled by studying mitochondrial protein import appear to be of general interest for investigations on intracellular protein sorting.

Biological Transport↗
PubMed1988

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.

Amino Acid Sequence↗
PubMed1988

The processing peptidase of yeast mitochondria: the two co-operating components MPP and PEP are structurally related.

Two proteins co-operate in the proteolytic cleavage of mitochondrial precursor proteins: the mitochondrial processing peptidase (MPP) and the processing enhancing protein (PEP). In order to understand the structure and function of this novel peptidase, we have isolated mutants of Saccharomyces cerevisiae which were temperature sensitive in the processing of mitochondrial precursor proteins. Here we report on the mif2 mutation which is deficient in MPP. Mitochondria from the mif2 mutant were able to import precursor proteins, but not to cleave the presequences. The MPP gene was isolated. MPP is a hydrophilic protein consisting of 482 amino acids. Notably, MPP exhibits remarkable sequence similarity to PEP. We speculate that PEP and MPP have a common origin and have evolved into two components with different but mutually complementing functions in processing of precursor proteins.

Amino Acid Sequence↗
PubMed1987

Successive translocation into and out of the mitochondrial matrix: targeting of proteins to the intermembrane space by a bipartite signal peptide.

We investigated the import and sorting pathways of cytochrome b2 and cytochrome c1, which are functionally located in the intermembrane space of mitochondria. Both proteins are synthesized on cytoplasmic ribosomes as larger precursors and are processed in mitochondria in two steps upon import. The precursors are first translocated across both mitochondrial membranes via contact sites into the matrix. Processing by the matrix peptidase leads to intermediate-sized forms, which are subsequently redirected across the inner membrane. The second proteolytic processing occurs in the intermembrane space. We conclude that the hydrophobic stretches in the presequences of the intermediate-sized forms do not stop transfer across the inner membrane, but rather act as transport signals to direct export from the matrix into the intermembrane space.

Cytochrome c Group↗
PubMed1987

Mitochondrial precursor proteins are imported through a hydrophilic membrane environment.

We have analyzed how translocation intermediates of imported mitochondrial precursor proteins, which span contact sites, interact with the mitochondrial membranes. F1-ATPase subunit beta (F1 beta) was trapped at contact sites by importing it into Neurospora mitochondria in the presence of low levels of nucleoside triphosphates. This F1 beta translocation intermediate could be extracted from the membranes by treatment with protein denaturants such as alkaline pH or urea. By performing import at low temperatures, the ADP/ATP carrier was accumulated in contact sites of Neurospora mitochondria and cytochrome b2 in contact sites of yeast mitochondria. These translocation intermediates were also extractable from the membranes at alkaline pH. Thus, translocation of precursor proteins across mitochondrial membranes seems to occur through an environment which is accessible to aqueous perturbants. We propose that proteinaceous structures are essential components of a translocation apparatus present in contact sites.

Intracellular Membranes↗
PubMed1987

Integral membrane polypeptides of rat liver peroxisomes: topology and response to different metabolic states.

Rats were treated with clofibrate, a hypolipidemic drug, and with thyroxine. Both drugs which are known to cause peroxisome proliferation, and a concomitant increase in peroxisomal fatty acid beta-oxidation activity in liver increased one of the major integral peroxisomal membrane polypeptides (PMPs), with apparent molecular mass of 69-kDa, six- and twofold, respectively. On the other hand hypothyroidism caused a decrease in peroxisomal fatty acid beta-oxidation activity and considerably lowered the concentration of PMP 69 in the peroxisomal membrane. Two other PMPs with apparent molecular masses of 36 and 22 kDa were not influenced by these treatments. The PMPs with apparent molecular masses of 42, 28, and 26 kDa were shown to be derived from the 69-kDa polypeptide by the activity of a yet uncharacterized endogenous protease during isolation of peroxisomes. Limited proteolysis of intact peroxisomes using proteinase K and subtilisin further substantiated that some portion of the 69-kDa polypeptide extends into the cytoplasm. The 36- and the 22-kDa polypeptides were accessible to proteolytic attack to a much lower extent and, therefore, are supposed to be rather deeply embedded within the peroxisomal membrane. It is demonstrated that peroxisomal acyl-CoA synthetase, an integral PMP extending partially into the cytoplasm, and PMP 69 are not identical polypeptides. Comparison of the peroxisomal membrane with that of mitochondria and microsomes revealed that the 69- and 22-kDa polypeptides as well as the bifunctional protein of the peroxisomal fatty acid beta-oxidation pathway were specifically located only in peroxisomes. Considerable amounts of a polypeptide cross-reacting with the antiserum against the 36-kDa polypeptide were found in mitochondria.

Animals↗
PubMed1986

Transport into mitochondria and intramitochondrial sorting of the Fe/S protein of ubiquinol-cytochrome c reductase.

The Fe/S protein of complex III is encoded by a nuclear gene, synthesized in the cytoplasm as a precursor with a 32 residue amino-terminal extension, and transported to the outer surface of the inner mitochondrial membrane. Our data suggest the following transport pathway. First, the precursor is translocated via translocation contact sites into the matrix. There, cleavage to an intermediate containing an eight residue extension occurs. The intermediate is then redirected across the inner membrane, processed to the mature subunit, and assembled into complex III. We suggest that the folding and membrane-translocation pathway in the endosymbiotic ancestor of mitochondria has been conserved during evolution of eukaryotic cells; transfer of the gene for Fe/S protein to the nucleus has led to addition of the presequence, which routes the precursor back to its "ancestral" assembly pathway.

Amino Acid Sequence↗
PubMed1985

Improved isolation and purification of rat liver peroxisomes by combined rate zonal and equilibrium density centrifugation.

Two different peroxisome preparations were isolated from male rat liver by using total homogenate (TH) as the starting material for one and the light mitochondrial (L) fraction for the other. The technique worked out is based on rate zonal (RZ) centrifugation in a sucrose gradient and subsequent isopycnic centrifugation in a Nycodenz gradient. The peroxisome fraction isolated from the L fraction consisted of 97-98% peroxisomal protein with catalase activity 49-fold enriched over TH. The peroxisome preparation isolated directly from TH represented about 55% of the total liver peroxisome population and had catalase activity 43-fold enriched compared with TH. The contribution of peroxisome protein to the liver protein was calculated to be in the range 1.82-2.02%. Peroxisomes isolated from TH were considerably more heterogeneous in size than peroxisomes isolated from the L fraction. Comparison of the polypeptide patterns of both preparations by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed some quantitative differences. Several major polypeptides were found to be exclusively located in the peroxisome membrane. These polypeptides migrated in the gel with apparent molecular masses of 69, 42.5, 36, 26, 21, and 15 kDa.

Animals↗
PubMed1983

Rat liver peroxisomes, II. Stimulation of peroxisomal fatty-acid beta-oxidation by thyroid hormones.

From liver homogenates of control and thyroxine-treated rats the light mitochondrial fractions were prepared by differential centrifugation. Mitochondria and peroxisomes of these fractions were further separated by isopycnic sucrose density gradient centrifugation. Analysis of the activities of the peroxisomal marker enzymes catalase and urate oxidase throughout the gradient exhibited completely corresponding distributions with that of peroxisomal palmitoyl-CoA oxidation. Thyroxine treatment did not change the proportion of mitochondria and peroxisomes present in the differential fractions of the homogenate. Consequently, the activities of peroxisomal beta-oxidation in the light mitochondrial fractions of control and thyroxine-treated animals were directly comparable with each other. A 2.5-fold increase in peroxisomal palmitoyl-CoA oxidation raising the activity from 0.089 to 0.220 U/g as estimated in the light mitochondrial fractions was found following treatment of male rats with daily doses of 25 micrograms L-thyroxine (s.c.) over a period of 10 days. Total hepatic peroxisomal fatty-acyl-CoA oxidation was calculated based on estimations in the light mitochondrial fractions. The difficulties encountered herein are discussed. The present result replenishes the broad spectrum of cellular effects exerted by thyroid hormones and indicates that both the mitochondrial and the peroxisomal system contribute to the well known hormone effects such as increase in fatty-acid oxidation, O2 consumption and heat production.

Acyl Coenzyme A↗
PubMed1982

Rat liver peroxisomes. I. New peroxisome population induced by thyroid hormones in the liver of male rats.

The paper presents results that a hyperthyroid state for 5 to 10 days induces a new peroxisome population in the liver of male rats. This assumption is based on the following observations: 1. Treatment of male rats for five consecutive days with 25 micrograms thyroxine per day resulted in an increase of catalase and D-amino acid oxidase activities by 32 and 61%, respectively. On the other hand, urate oxidase activity declined by 20%. Prolonged treatment for ten days raised catalase activity by 68% and lowered urate oxidase activity by 35%. 2. Morphological examinations on the light and electron microscopic level revealed that the increase in peroxisomal enzyme activities was accompanied by an augmentation of peroxisomal profiles. "Microperoxisomes" and peroxisomes lacking the electron dense core were frequently observed. 3. The peroxisomes of the livers of thyroxine-treated animals exhibited an altered centrifugal behaviour in sucrose density gradients. The mean equilibrium density of normal liver peroxisomes was 1.23, whereas peroxisomes of thyroxine-treated livers equilibrated at the density of 1.20. It is discussed that peroxisomal metabolism may contribute considerably to the late biochemical effects of thyroid hormones, that means enhancement of lipid oxidation and heat production. Up to now these late effects were attributed to a stimulated mitochondrial metabolism only.

Animals↗