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At least 1,009 records · Page 56Linked to original sources
PubMed1999

Pex19p interacts with Pex3p and Pex10p and is essential for peroxisome biogenesis in Pichia pastoris.

We report the cloning and characterization of Pichia pastoris PEX19 by complementation of a peroxisome-deficient mutant strain. Import of peroxisomal targeting signal 1- and 2-containing peroxisomal matrix proteins is defective in pex19 mutants. PEX19 encodes a hydrophilic 299-amino acid protein with sequence similarity to Saccharomyces cerevisiae Pex19p and human and Chinese hamster PxF, all farnesylated proteins, as well as hypothetical proteins from Caenorhabditis elegans and Schizosaccharomyces pombe. The farnesylation consensus is conserved in PpPex19p but dispensable for function and appears unmodified under the conditions tested. Pex19p localizes predominantly to the cytosolic fraction. Biochemical and two-hybrid analyses confirmed that Pex19p interacts with Pex3p, as seen in S. cerevisiae, but unexpectedly also with Pex10p. Two-hybrid analysis demonstrated that the amino-terminal 42 amino acids of Pex19p interact with the carboxyl-terminal 335 amino acids of Pex3p. In addition, the extreme carboxyl terminus of Pex19p (67 amino acids) is required for interaction with the amino-terminal 380 amino acids of Pex10p. Biochemical and immunofluorescence microscopy analyses of pex19Delta cells identified the membrane protein Pex3p in peroxisome remnants that were not previously observed in S. cerevisiae. These small vesicular and tubular (early) remnants are morphologically distinct from other Pppex mutant (late) remnants, suggesting that Pex19p functions at an early stage of peroxisome biogenesis.

ATP-Binding Cassette Transporters↗
PubMed2000

Rab7: a key to lysosome biogenesis.

The molecular machinery behind lysosome biogenesis and the maintenance of the perinuclear aggregate of late endocytic structures is not well understood. A likely candidate for being part of this machinery is the small GTPase Rab7, but it is unclear whether this protein is associated with lysosomes or plays any role in the regulation of the perinuclear lysosome compartment. Previously, Rab7 has mainly been implicated in transport from early to late endosomes. We have now used a new approach to analyze the role of Rab7: transient expression of Enhanced Green Fluorescent Protein (EGFP)-tagged Rab7 wt and mutant proteins in HeLa cells. EGFP-Rab7 wt was associated with late endocytic structures, mainly lysosomes, which aggregated and fused in the perinuclear region. The size of the individual lysosomes as well as the degree of perinuclear aggregation increased with the expression levels of EGFP-Rab7 wt and, more dramatically, the active EGFP-Rab7Q67L mutant. In contrast, upon expression of the dominant-negative mutants EGFP-Rab7T22N and EGFP-Rab7N125I, which localized mainly to the cytosol, the perinuclear lysosome aggregate disappeared and lysosomes, identified by colocalization of cathepsin D and lysosome-associated membrane protein-1, became dispersed throughout the cytoplasm, they were inaccessible to endocytosed molecules such as low-density lipoprotein, and their acidity was strongly reduced, as determined by decreased accumulation of the acidotropic probe LysoTracker Red. In contrast, early endosomes associated with Rab5 and the transferrin receptor, late endosomes enriched in the cation-independent mannose 6-phosphate receptor, and the trans-Golgi network, identified by its enrichment in TGN-38, were unchanged. These data demonstrate for the first time that Rab7, controlling aggregation and fusion of late endocytic structures/lysosomes, is essential for maintenance of the perinuclear lysosome compartment.

Amino Acid Substitution↗
PubMed2004

The Oxa2 protein of Neurospora crassa plays a critical role in the biogenesis of cytochrome oxidase and defines a ubiquitous subbranch of the Oxa1/YidC/Alb3 protein family.

Proteins of the Oxa1/YidC/Alb3 family mediate the insertion of proteins into membranes of mitochondria, bacteria, and chloroplasts. Here we report the identification of a second gene of the Oxa1/YidC/Alb3 family in the genome of Neurospora crassa, which we have named oxa2. Its gene product, Oxa2, is located in the inner membrane of mitochondria. Deletion of the oxa2 gene caused a specific defect in the biogenesis of cytochrome oxidase and resulted in induction of the alternative oxidase (AOD), which bypasses the need for complex IV of the respiratory chain. The Oxa2 protein of N. crassa complements Cox18-deficient yeast mutants suggesting a common function for both proteins. The oxa2 sequence allowed the identification of a new subfamily of Oxa1/YidC/Alb3 proteins whose members appear to be ubiquitously present in mitochondria of fungi, plants, and animals including humans.

Amino Acid Sequence↗
PubMed2004

Oncostatin M-stimulated apical plasma membrane biogenesis requires p27(Kip1)-regulated cell cycle dynamics.

Oncostatin M regulates membrane traffic and stimulates apicalization of the cell surface in hepatoma cells in a protein kinase A-dependent manner. Here, we show that oncostatin M enhances the expression of the cyclin-dependent kinase (cdk)2 inhibitor p27(Kip1), which inhibits G(1)-S phase progression. Forced G(1)-S-phase transition effectively renders presynchronized cells insensitive to the apicalization-stimulating effect of oncostatin M. G(1)-S-phase transition prevents oncostatin M-mediated recruitment of protein kinase A to the centrosomal region and precludes the oncostatin M-mediated activation of a protein kinase A-dependent transport route to the apical surface, which exits the subapical compartment (SAC). This transport route has previously been shown to be crucial for apical plasma membrane biogenesis. Together, our data indicate that oncostatin M-stimulated apicalization of the cell surface is critically dependent on the ability of oncostatin M to control p27(Kip1)/cdk2-mediated G(1)-S-phase progression and suggest that the regulation of apical plasma membrane-directed traffic from SAC is coupled to centrosome-associated signaling pathways.

Cell Compartmentation↗
PubMed2004

Normal biogenesis and cycling of empty synaptic vesicles in dopamine neurons of vesicular monoamine transporter 2 knockout mice.

The neuronal isoform of vesicular monoamine transporter, VMAT2, is responsible for packaging dopamine and other monoamines into synaptic vesicles and thereby plays an essential role in dopamine neurotransmission. Dopamine neurons in mice lacking VMAT2 are unable to store or release dopamine from their synaptic vesicles. To determine how VMAT2-mediated filling influences synaptic vesicle morphology and function, we examined dopamine terminals from VMAT2 knockout mice. In contrast to the abnormalities reported in glutamatergic terminals of mice lacking VGLUT1, the corresponding vesicular transporter for glutamate, we found that the ultrastructure of dopamine terminals and synaptic vesicles in VMAT2 knockout mice were indistinguishable from wild type. Using the activity-dependent dyes FM1-43 and FM2-10, we also found that synaptic vesicles in dopamine neurons lacking VMAT2 undergo endocytosis and exocytosis with kinetics identical to those seen in wild-type neurons. Together, these results demonstrate that dopamine synaptic vesicle biogenesis and cycling are independent of vesicle filling with transmitter. By demonstrating that such empty synaptic vesicles can cycle at the nerve terminal, our study suggests that physiological changes in VMAT2 levels or trafficking at the synapse may regulate dopamine release by altering the ratio of fillable-to-empty synaptic vesicles, as both continue to cycle in response to neural activity.

Animals↗
PubMed2005

Targeting of Tsc13p to nucleus-vacuole junctions: a role for very-long-chain fatty acids in the biogenesis of microautophagic vesicles.

TSC13 is required for the biosynthesis of very-long-chain fatty acids (VLCFAs) in yeast. Tsc13p is a polytopic endoplasmic reticulum (ER) membrane protein that accumulates at nucleus-vacuole (NV) junctions, which are formed through Velcro-like interactions between Nvj1p in the perinuclear ER and Vac8p on the vacuole membrane. NV junctions mediate piecemeal microautophagy of the nucleus (PMN), during which bleb-like portions of the nucleus are extruded into invaginations of the vacuole membrane and degraded in the vacuole lumen. We report that Tsc13p is sequestered into NV junctions from the peripheral ER through Vac8p-independent interactions with Nvj1p. During nutrient limitation, Tsc13p is incorporated into PMN vesicles in an Nvj1p-dependent manner. The lumenal diameters of PMN blebs and vesicles are significantly reduced in tsc13-1 and tsc13-1 elo3-Delta mutant cells. PMN structures are also smaller in cells treated with cerulenin, an inhibitor of de novo fatty acid synthesis and elongation. The targeting of Tsc13p-GFP into NV junctions is perturbed by cerulenin, suggesting that its binding to Nvj1p depends on the availability of fatty acid substrates. These results indicate that Nvj1p retains and compartmentalizes Tsc13p at NV junctions and that VLCFAs contribute to the normal biogenesis of trilaminar PMN structures in yeast.

Acylation↗
PubMed2006

Mitochondria-associated yeast mRNAs and the biogenesis of molecular complexes.

The coherence of mitochondrial biogenesis relies on spatiotemporally coordinated associations of 800-1000 proteins mostly encoded in the nuclear genome. We report the development of new quantitative analyses to assess the role of local protein translation in the construction of molecular complexes. We used real-time PCR to determine the cellular location of 112 mRNAs involved in seven mitochondrial complexes. Five typical cases were examined by an improved FISH protocol. The proteins produced in the vicinity of mitochondria (MLR proteins) were, almost exclusively, of prokaryotic origin and are key elements of the core construction of the molecular complexes; the accessory proteins were translated on free cytoplasmic polysomes. These two classes of proteins correspond, at least as far as intermembrane space (IMS) proteins are concerned, to two different import pathways. Import of MLR proteins involves both TOM and TIM23 complexes whereas non-MLR proteins only interact with the TOM complex. Site-specific translation loci, both outside and inside mitochondria, may coordinate the construction of molecular complexes composed of both nuclearly and mitochondrially encoded subunits.

Carrier Proteins↗
PubMed1999

Impairment of peroxisomal biogenesis in human colon carcinoma.

Peroxisomes and the activities of their enzymes have been reported to be significantly reduced in various types of tumors including the colon carcinoma. Therefore, the present study was designed to investigate the gene expression of several peroxisomal proteins in human colon carcinoma and additionally those of the peroxisome proliferator activated receptor alpha (PPARalpha) and PEX5, a receptor protein involved in the import of most peroxisomal matrix proteins. Samples from adenocarcinomas and adjacent normal colon were analyzed by immunohistochemistry and western blotting. The mRNA content was assessed by a novel sensitive dot blot RNase protection assay and northern blotting. By immunohistochemistry, peroxisomes were distinctly visualized in normal colonocytes but were not detected in colon carcinoma cells. The protein levels of catalase (CAT), acyl-CoA oxidase as well as the 22 and 70 kDa peroxisomal membrane proteins (PMP22 and PMP70) were all significantly decreased in carcinomas. The corresponding mRNAs for CAT and PMP70, however, were unchanged. In contrast, the mRNA of PEX5 was significantly increased. The expression of PPARalpha was not altered in tumors, neither at protein nor mRNA levels. These observations show that the reduction of peroxisomes and their proteins in colon carcinoma is not due to a generalized reduction of transcription of their genes. It seems more likely that this phenomenon is regulated at a post-transcriptional or translational level. Alternatively, and more likely, an impairment of the biogenesis of the organelle could account for the paucity of peroxisomes in colon carcinoma.

ATP-Binding Cassette Transporters↗
PubMed2004

A new role for the von Hippel-Lindau tumor suppressor protein: stimulation of mitochondrial oxidative phosphorylation complex biogenesis.

Although mitochondrial deficiency in cancer has been described by Warburg, many years ago, the mechanisms underlying this impairment remain essentially unknown. Many types of cancer cells are concerned and, in particular, clear cell renal carcinoma (CCRC). In this cancer, the tumor suppressor gene, VHL (von Hippel-Lindau factor) is invalidated. Previous studies have shown that the transfection of the VHL gene in VHL-deficient cells originating from CCRCs could suppress their ability to form tumors when they were injected into nude mice. However, various additional genetic alterations are observed in such cancer cells. In order to investigate whether VHL invalidation was related to the mitochondrial impairment, we have studied the effects of wild-type VHL transfection into VHL-deficient 786-0 or RCC10 cells on their oxidative phosphorylation (OXPHOS) subunit contents and functions. We show that the presence of wild-type VHL protein (pVHL) increased mitochondrial DNA and respiratory chain protein contents and permitted the cells to rely on their mitochondrial ATP production to grow in the absence of glucose. In parallel to mtDNA increase, the presence of pVHL up regulated the mitochondrial transcription factor A, as shown by western blot analysis. In conclusion, in CCRCs, pVHL deficiency is one of the factors responsible for down-regulation of the biogenesis of OXPHOS complexes.

Adenosine Triphosphate↗
PubMed1997

The Hansenula polymorpha PEX14 gene encodes a novel peroxisomal membrane protein essential for peroxisome biogenesis.

We have cloned the Hansenula polymorpha PEX14 gene by functional complementation of the chemically induced pex14-1 mutant, which lacked normal peroxisomes. The sequence of the PEX14 gene predicts a novel protein product (Pex14p) of 39 kDa which showed no similarity to any known protein and lacked either of the two known peroxisomal targeting signals. Biochemical and electron microscopical analysis indicated that Pex14p is a component of the peroxisomal membrane. The synthesis of Pex14p is induced by peroxisome-inducing growth conditions. In cells of both pex14-1 and a PEX14 disruption mutant, peroxisomal membrane remnants were evident; these contained the H.polymorpha peroxisomal membrane protein Pex3p together with a small amount of the major peroxisomal matrix proteins alcohol oxidase, catalase and dihydroxyacetone synthase, the bulk of which resided in the cytosol. Unexpectedly, overproduction of Pex14p in wild-type H. polymorpha cells resulted in a peroxisome-deficient phenotype typified by the presence of numerous small vesicles which lacked matrix proteins; these were localized in the cytosol. Apparently, the stoichiometry of Pex14p relative to one or more other components of the peroxisome biogenesis machinery appears to be critical for protein import.

Amino Acid Sequence↗
PubMed1997

Altered expression of a novel adaptin leads to defective pigment granule biogenesis in the Drosophila eye color mutant garnet.

Drosophila eye pigmentation defects have thus far been attributed to mutations in genes encoding enzymes required for biosynthesis of pigments and to ABC-type membrane transporters for pigments or their precursors. We report here that a defect in a gene encoding a putative coat adaptor protein leads to the eye color defect of garnet mutants. We first identified a human cDNA encoding delta-adaptin, a structural homolog of the alpha- and gamma-adaptin subunits of the clathrin coat adaptors AP-1 and AP-2, respectively. Biochemical analyses demonstrated that delta-adaptin is a component of the adaptor-like complex AP-3 in human cells. We then isolated a full-length cDNA encoding the Drosophila ortholog of delta-adaptin and found that transcripts specified by this cDNA are altered in garnet mutant flies. Examination by light and electron microscopy indicated that these mutant flies have reduced numbers of eye pigment granules, which correlates with decreased levels of both pteridine (red) and ommachrome (brown) pigments. Thus, the eye pigmentation defect in the Drosophila garnet mutant may be attributed to compromised function of a coat protein involved in intracellular transport processes required for biogenesis or function of pigment granules.

Adaptor Protein Complex alpha Subunits↗
PubMed1998

A dynamin-like protein in Arabidopsis thaliana is involved in biogenesis of thylakoid membranes.

Dynamin, a GTP-binding protein found in rat brain, plays a role in endocytosis. Suborganellar fractionation studies of Arabidopsis leaf tissue revealed that a dynamin-like protein, ADL1, is localized in the thylakoid membranes of chloroplasts. This notion was supported further by in vivo targeting experiments using an ADL1-green fluorescent fusion protein and immunogold labeling with the anti-ADL1 antibody. Transgenic plants harboring various deletion mutant genes of ADL1 had a yellow leaf phenotype where the cells had very few chloroplasts. In addition, the remaining chloroplasts appeared morphologically not fully developed. The detailed structure of the chloroplasts revealed by electron microscopy showed a greatly reduced amount of thylakoid membranes. Also, the level of thylakoid membrane proteins such as the light-harvesting complex II and CP29 was greatly reduced in these transgenic plants. When we examined the expression of the ADL1 deletion mutant genes, these genes were highly expressed at the transcriptional level. However, the mutant ADL1s were not detectable at the protein level by Western blot analysis. Moreover, the endogenous ADL1 protein level was greatly reduced in these transgenic plants, probably due to a post-transcriptional silencing effect of the transgenes. We propose, therefore, that ADLl is involved in the biogenesis of thylakoid membranes.

Arabidopsis↗
PubMed1998

Biogenesis of Tim23 and Tim17, integral components of the TIM machinery for matrix-targeted preproteins.

We analysed the import pathway of Tim23 and of Tim17, components of the mitochondrial import machinery for matrix-targeted preproteins. Tim23 contains two independent import signals. One is located within the first 62 amino acid residues of the hydrophilic domain that, in the assembled protein, is exposed to the intermembrane space. This signal mediates translocation of Tim23 across the outer membrane independently of the membrane potential, DeltaPsi. A second import signal is located in the C-terminal membrane-integrated portion of Tim23. It mediates translocation across the outer membrane and insertion into the inner membrane in a strictly DeltaPsi-dependent fashion. Structurally, Tim17 is related to Tim23 but lacks a hydrophilic domain. It contains an import signal in the C-terminal half and its import requires DeltaPsi. The DeltaPsi-dependent import signals of Tim23 and Tim17 are located at corresponding sites in these two homologous proteins. They exhibit features reminiscent of the positively charged N-terminal presequences of matrix-targeted precursors. Import of Tim23 and its insertion into the inner membrane requires Tim22 but not functional Tim23. Thus, biogenesis of the Tim23.17 complex depends on the Tim22 complex, which is the translocase identified as mediating the import of carrier proteins.

Amino Acid Sequence↗
PubMed1998

Ramifications of kinetic partitioning on usher-mediated pilus biogenesis.

The biogenesis of diverse adhesive structures in a variety of Gram-negative bacterial species is dependent on the chaperone/usher pathway. Very little is known about how the usher protein translocates protein subunits across the outer membrane or how assembly of these adhesive structures occurs. We have discovered several mechanisms by which the usher protein acts to regulate the ordered assembly of type 1 pili, specifically through critical interactions of the chaperone-adhesin complex with the usher. A study of association and dissociation events of chaperone-subunit complexes with the usher in real time using surface plasmon resonance revealed that the chaperone-adhesin complex has the tightest and fastest association with the usher. This suggests that kinetic partitioning of chaperone-adhesin complexes to the usher is a defining factor in tip localization of the adhesin in the pilus. Furthermore, we identified and purified a chaperone-adhesin-usher assembly intermediate that was formed in vivo. Trypsin digestion assays showed that the usher in this complex was in an altered conformation, which was maintained during pilus assembly. The data support a model in which binding of the chaperone-adhesin complex to the usher stabilizes the usher in an assembly-competent conformation and allows initiation of pilus assembly.

Adhesins, Bacterial↗
PubMed1999

The Hdj-2/Hsc70 chaperone pair facilitates early steps in CFTR biogenesis.

The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride ion channel constructed from two membrane-spanning domains (MSDs), two nucleotide-binding domains (NBD) and a regulatory (R) domain. The NBDs and R-domain are cytosolic and how they are assembled with the MSDs to achieve the native CFTR structure is not clear. Human DnaJ 2 (Hdj-2) is a co-chaperone of heat shock cognate 70 (Hsc70) which is localized to the cytosolic face of the ER. Whether Hdj-2 directs Hsc70 to facilitate the assembly of cytosolic regions on CFTR was investigated. We report that immature ER forms of CFTR and DeltaF508 CFTR can be isolated in complexes with Hdj-2 and Hsc70. The DeltaF508 mutation is localized in NBD1 and causes the CFTR to misfold. Levels of complex formation between DeltaF508 CFTR and Hdj-2/Hsp70 were approximately 2-fold higher than those with CFTR. The earliest stage at which Hdj-2/Hsc70 could bind CFTR translation intermediates coincided with the expression of NBD1 in the cytosol. Interestingly, complex formation between Hdj-2 and nascent CFTR was greatly reduced after expression of the R-domain. In experiments with purified components, Hdj-2 and Hsc70 acted synergistically to suppress NBD1 aggregation. Collectively, these data suggest that Hdj-2 and Hsc70 facilitate early steps in CFTR assembly. A putative step in the CFTR folding pathway catalyzed by Hdj-2/Hsc70 is the formation of an intramolecular NBD1-R-domain complex. Whether this step is defective in the biogenesis of DeltaF508 CFTR will be discussed.

Carrier Proteins↗
PubMed2000

Stress induces peroxisome biogenesis genes.

Peroxisomes are the cellular location of many antioxidants and are themselves significant producers of reactive oxygen species. In this report we demonstrate the induction of peroxisome biogenesis genes in both plant and animal cells by the universal stress signal molecule hydrogen peroxide. Using PEX1-LUC transgenic plants, rapid local and systemic induction of PEX1-luciferase could be demonstrated in vivo in response to physiological levels of hydrogen peroxide. PEX1-luciferase was also induced in response to wounding and to infection with an avirulent pathogen. We propose a model in which various stress situations that lead to the production of hydrogen peroxide can be ameliorated by elaboration of the peroxisome compartment to assist in restoration of the cellular redox balance.

ATPases Associated with Diverse Cellular Activitie↗
PubMed2001

A nuclear AAA-type ATPase (Rix7p) is required for biogenesis and nuclear export of 60S ribosomal subunits.

Ribosomal precursor particles are assembled in the nucleolus before export into the cytoplasm. Using a visual assay for nuclear accumulation of 60S subunits, we have isolated several conditional-lethal strains with defects in ribosomal export (rix mutants). Here we report the characterization of a mutation in an essential gene, RIX7, which encodes a novel member of the AAA ATPase superfamily. The rix7-1 temperature-sensitive allele carries a point mutation that causes defects in pre-rRNA processing, biogenesis of 60S ribosomal subunits, and their subsequent export into the cytoplasm. Rix7p, which associates with 60S ribosomal precursor particles, localizes throughout the nucleus in exponentially growing cells, but concentrates in the nucleolus in stationary phase cells. When cells resume growth upon shift to fresh medium, Rix7p-green fluorescent protein exhibits a transient perinuclear location. We propose that a nuclear AAA ATPase is required for restructuring nucleoplasmic 60S pre-ribosomal particles to make them competent for nuclear export.

Adenosine Triphosphatases↗
PubMed2002

Dynamin and clathrin are required for the biogenesis of a distinct class of secretory vesicles in yeast.

Yeast produce two classes of secretory vesicles (SVs) that differ in both density and cargo protein content. In late-acting secretory mutants (e.g. snc1(ala43) and sec6-4), both low- (LDSV) and high-density (HDSV) classes of vesicles accumulate at restrictive temperatures. Here, we have found that disruptions in the genes encoding a dynamin-related protein (VPS1) or clathrin heavy chain (CHC1) abolish HDSV production, yielding LDSVs that contain all secreted cargos. Interestingly, disruption of the PEP12 gene, which encodes the t-SNARE that mediates all Golgi to pre-vacuolar compartment (PVC) transport, also abolishes HDSV production. In contrast, deletions in genes that selectively confer vacuolar hydrolase sorting to the PVC or protein transport to the vacuole (i.e. VPS34 and VAM3, respectively) have no effect. Thus, one branch of the secretory pathway in yeast involves an intermediate sorting compartment and has a specific requirement for clathrin and a dynamin-related protein in SV biogenesis.

Carrier Proteins↗