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PubMed1987

Trigger factor: a soluble protein that folds pro-OmpA into a membrane-assembly-competent form.

Pro-OmpA that is synthesized in vitro can assemble into bacterial inner membrane vesicles in the presence of ATP and NADH. We have purified pro-OmpA to determine which additional soluble proteins are necessary for its membrane assembly. [35S]Pro-OmpA was bound to Sepharose-linked antibody to OmpA, then eluted with 8 M urea and chromatographed on an anion-exchange resin in 8 M urea. This pro-OmpA is purified 2000-fold and is radiochemically pure. After dialysis, it is soluble but incompetent for membrane assembly. Addition of an Escherichia coli cytoplasmic fraction (S100) to the assembly reaction does not allow translocation. However, when S100 is added to pro-OmpA prior to dialysis, full assembly competence is restored, suggesting that a soluble factor, termed "trigger factor," triggers the folding of pro-OmpA into an assembly-competent form as the urea is removed. We noted that, prior to the last purification step, the immunoaffinity-purified pro-OmpA was partially competent for membrane assembly without addition of trigger factor. To test whether trigger factor had bound to the antibody column by means of its association with pro-OmpA, the crude pro-OmpA was acid-denatured prior to immunoadsorption. In this experiment, the trigger factor did not bind to the anti-OmpA column, and S100 was required for renaturation of this [35S]pro-OmpA. As suggested by this experiment, the crude [35S]pro-OmpA was in a complex with other proteins. Sedimentation velocity studies showed that the trigger factor has an apparent molecular weight of approximately 60,000. We propose that it is required for translocation-competent folding of pro-OmpA and other precursor proteins.

Adenosine Triphosphate↗
PubMed1987

Self-assembly of proglycinin and hybrid proglycinin synthesized in vitro from cDNA.

An in vitro system was developed that results in the self-assembly of subunit precursors into complexes that resemble those found naturally in the endoplasmic reticulum. Subunits of glycinin, the predominant seed protein of soybeans, were synthesized from modified cDNAs using a combination of the SP6 transcription and the rabbit reticulocyte translation systems. Subunits produced from plasmid constructions that encoded either Gy4 or Gy5 gene products, but modified such that their signal sequences were absent, self-assembled into trimers equivalent in size to those precursors found in the endoplasmic reticulum. In contrast, proteins synthesized in vitro from Gy4 constructs failed to self-assemble when the signal sequence was left intact (e.g., preproglycinin) or when the coding sequence was modified to remove 27 amino acids from an internal hydrophobic region, which is highly conserved among the glycinin subunits. Various hybrid subunits were also produced by trading portions of Gy4 and Gy5 cDNAs and all self-assembled in our system. The in vitro assembly system provides an opportunity to study the self-assembly of precursors and to probe for regions important for assembly. It will also be helpful in attempts to engineer beneficial nutritional changes into this important food protein.

Journal Article↗
PubMed1994

Expression of tobacco mosaic virus coat protein and assembly of pseudovirus particles in Escherichia coli.

The bidirectional self-assembly of tobacco mosaic virus (TMV, common or U1 strain) has been studied extensively in vitro. Foreign single-stranded RNA molecules containing the TMV origin-of-assembly sequence (OAS, 75-432 nt in length) are also packaged by TMV coat protein (CP) in vitro to form helical pseudovirus particles. To study virus assembly in vivo requires an easily manipulated model system, independent of replication in plants. The TMV assembly machinery also provides a convenient means to protect and recover chimeric gene transcripts of almost any length or sequence for a variety of applications. Native TMV CP expressed in and purified from Escherichia coli formed nonhelical, stacked aggregates after dialysis into pH 5 buffer and was inactive for in vitro assembly with TMV RNA. U1 CP derivatives in which the second amino acid was changed from Ser to Ala or Pro, nonacetylated N termini found in two natural strains of the virus, failed to remediate these anomalous properties. However, in vivo coexpression of CP and single-stranded RNAs (up to approximately 2 kb) containing the TMV OAS gave high yields of helical pseudovirus particles of the predicted length (up to 7.4 +/- 1.4 micrograms/mg of total bacterial protein). If the OAS-containing RNA was first recruited into bacterial polyribosomes, elongation of pseudovirus assembly was blocked. In vivo, E. coli expression of a full-length cDNA clone of the TMV genome (6.4 kb) resulted in high, immunodetectable levels of CP and assembly of sufficient intact genomic RNA to initiate systemic infection of susceptible tobacco plants.

Base Sequence↗
PubMed1996

In vitro insertion and assembly of outer membrane protein PhoE of Escherichia coli K-12 into the outer membrane. Role of Triton X-100.

The assembly of the in vitro synthesized outer membrane protein PhoE into purified outer membranes was investigated. The assembly appeared to be strongly stimulated by the presence of low amounts of Triton X-100 (optimal 0.08%, w/v). The role of Triton X-100 in the in vitro system was further examined. Pretreating outer membranes with Triton X-100 did not make the membranes competent for correct assembly, indicating that the detergent did not act on the membrane but at the protein level. PhoE became assembly-incompetent with a half-life of approximately 12 min and 90 s at 37 degrees C in the absence and presence, respectively, of 0.08% Triton X-100. Apparently, Triton X-100 induces an assembly-competent state in the PhoE protein with a very short half-life. Furthermore, the efficiency of correct assembly of PhoE was greatly reduced when outer membranes of deep rough lipopolysaccharide mutants were used, indicating an important role of lipopolysaccharides in the assembly of the porin.

Cell Membrane↗
PubMed1996

Inhibition of the microsomal triglyceride transfer protein blocks the first step of apolipoprotein B lipoprotein assembly but not the addition of bulk core lipids in the second step.

The microsomal triglyceride transfer protein (MTP) is required for assembly and secretion of the lipoproteins containing apolipoprotein B (apoB): very low density lipoproteins and chylomicrons. Evidence indicates that the subclasses of these lipoproteins that contain apoB-48 are assembled in a distinct two-step process; first a relatively lipid-poor primordial lipoprotein precursor is produced, and then bulk neutral lipids are added to form the core of these spherical particles. To determine if either step is mediated by MTP, a series of clonal cell lines stably expressing apoB-53 and MTP was established in non-lipoprotein-producing HeLa cells. MTP activity in these cells was approximately 30%, and apoB secretion was 7-33% of that in HepG2 cells on a molar basis. Despite having robust levels of triglyceride and phospholipid synthesis, these cell lines, as exemplified by HLMB53-59, secreted >90% of the apoB-53 on relatively lipid-poor particles in the density range of 1.063-1.21 g/ml. These results suggested that coexpression of MTP and apoB only reconstituted the first but not the second step in lipoprotein assembly. To extend this observation, additional studies were carried out in McArdle RH-7777 rat hepatoma cells, in which the second step of apoB-48 lipoprotein assembly is well defined. Treatment of these cells with the MTP photoaffinity inhibitor BMS-192951 before pulse labeling with [35S]methionine/cysteine led to an 85% block of both apoB-48 and apoB-100 but not apoAI secretion, demonstrating inhibition of the first step of lipoprotein assembly. After a 30-min [35S]methioneine/cysteine pulse labeling and 120 min of chase, all of the nascent apoB-48 was observed to have a density of high density lipoproteins (1.063-1.21 g/ml), indicating that only the first step of lipoprotein assembly had occurred. The addition of oleic acid to the cell culture media activated the second step as evidenced by the conversion of the apoB-48 high density lipoproteins to very low density lipoproteins (d < 1.006 g/ml) during an extended chase period. Inactivation of MTP after completion of the first step, but before stimulation of the second step by the addition of oleic acid, did not block this conversion. Thus, inhibition of MTP did not hinder the addition of bulk core lipid to the primordial lipoprotein precursor particles, indicating that MTP is not required for the second step of apoB-48 lipoprotein assembly.

Animals↗
PubMed1998

Integrin-linked protein kinase regulates fibronectin matrix assembly, E-cadherin expression, and tumorigenicity.

Fibronectin (Fn) matrix plays important roles in many biological processes including morphogenesis and tumorigenesis. Recent studies have demonstrated a critical role of integrin cytoplasmic domains in regulating Fn matrix assembly, implying that intracellular integrin-binding proteins may be involved in controlling extracellular Fn matrix assembly. We report here that overexpression of integrin-linked kinase (ILK), a newly identified serine/threonine kinase that binds to the integrin beta1 cytoplasmic domain, dramatically stimulated Fn matrix assembly in epithelial cells. The integrin-linked kinase activity is involved in transducing signals leading to the up-regulation of Fn matrix assembly, as overexpression of a kinase-inactive ILK mutant failed to enhance the matrix assembly. Moreover, the increase in Fn matrix assembly induced by ILK overexpression was accompanied by a substantial reduction in the cellular E-cadherin. Finally, we show that ILK-overexpressing epithelial cells readily formed tumors in nude mice, despite forming an extensive Fn matrix. These results identify ILK as an important regulator of pericellular Fn matrix assembly, and suggest a novel critical role of this integrin-linked kinase in cell growth, cell survival, and tumorigenesis.

Amino Acid Sequence↗
PubMed1998

The role of DnaJ-like proteins in glucocorticoid receptor.hsp90 heterocomplex assembly by the reconstituted hsp90.p60.hsp70 foldosome complex.

The glucocorticoid receptor (GR) is recovered from hormone-free cells in a heterocomplex with the molecular chaperone hsp90, which is required to produce the proper folding state for steroid binding. GR.hsp90 heterocomplexes are formed by a multiprotein system that appears to exist in all eukaryotic cells. Recently, we have reconstituted a receptor.hsp90 heterocomplex assembly system with purified rabbit hsp90 and hsp70 and bacterially expressed human p23 and p60. We have shown that hsp90, p60, and hsp70 form an hsp90.p60. hsp70 complex that converts the GR from a non-steroid binding to a steroid binding form (Dittmar, K. D., and Pratt, W. B. (1997) J. Biol. Chem. 272, 13047-13054). The resulting GR.hsp90 heterocomplex rapidly disassembles unless p23 is present to bind to the ATP-dependent conformation of hsp90 and stabilize its association with the receptor (Dittmar, K. D., Demady, D. R., Stancato, L. F., Krishna, P., and Pratt, W. B. (1997) J. Biol. Chem. 272, 21213-21220). In the current work, we show that the purified rabbit hsp70 utilized in prior studies is contaminated with a small amount of the rabbit DnaJ homolog hsp40. Elimination of the hsp40 from the purified GR.hsp90 assembly system reduces assembly activity, and the activity is restored by addition of the purified yeast DnaJ homolog YDJ-1. hsp40 is a component of the hsp90.p60.hsp70 foldosome complex isolated from reticulocyte lysate with antibody against p60. Under conditions that promote binding of p23 to hsp90 (elevated temperature, ATP, Nonidet P-40, molybdate), a five-membered (p23. hsp90.p60.hsp70.hsp40) complex of chaperone proteins is formed in reticulocyte lysate or from purified proteins. The hsp40-free, purified assembly system has a modest level of assembly activity that is maximally potentiated by YDJ-1 when it is present at about one-twentieth the concentration of hsp70. Although hsp40 is not in the final GR.hsp90 heterocomplex isolated from L cell cytosol, it is in the GR.hsp90 heterocomplex assembled in reticulocyte lysate. We conclude that hsp40 is a component of the multiprotein hsp90-based chaperone system where it potentiates GR.hsp90 heterocomplex assembly.

Animals↗
PubMed1998

Impaired assembly of E1 decarboxylase of the branched-chain alpha-ketoacid dehydrogenase complex in type IA maple syrup urine disease.

The E1 decarboxylase component of the human branched-chain ketoacid dehydrogenase complex comprises two E1alpha (45.5 kDa) and two E1beta (37.5 kDa) subunits forming an alpha2 beta2 tetramer. In patients with type IA maple syrup urine disease, the E1alpha subunit is affected, resulting in the loss of E1 and branched-chain ketoacid dehydrogenase catalytic activities. To study the effect of human E1alpha missense mutations on E1 subunit assembly, we have developed a pulse-chase labeling protocol based on efficient expression and assembly of human (His)6-E1alpha and untagged E1beta subunits in Escherichia coli in the presence of overexpressed chaperonins GroEL and GroES. Assembly of the two 35S-labeled E1 subunits was indicated by their co-extraction with Ni2+-nitrilotriacetic acid resin. The nine E1alpha maple syrup urine disease mutants studied showed aberrant kinetics of assembly with normal E1beta in the 2-h chase compared with the wild type and can be classified into four categories of normal (N222S-alpha and R220W-alpha), moderately slow (G245R-alpha), slow (G204S-alpha, A240P-alpha, F364C-alpha, Y368C-alpha, and Y393N-alpha), and no (T265R-alpha) assembly. Prolonged induction in E. coli grown in the YTGK medium or lowering of induction temperature from 37 to 28 degreesC (in the case of T265R-alpha), however, resulted in the production of mutant E1 proteins. Separation of purified E1 proteins by sucrose density gradient centrifugation showed that the wild-type E1 existed entirely as alpha2 beta2 tetramers. In contrast, a subset of E1alpha missense mutations caused the occurrence of exclusive alphabeta dimers (Y393N-alpha and F364C-alpha) or of both alpha2beta2 tetramers and lower molecular weight species (Y368C-alpha and T265R-alpha). Thermal denaturation at 50 degreesC indicated that mutant E1 proteins aggregated more rapidly than wild type (rate constant, 0.19 min-1), with the T265R-alpha mutant E1 most severely affected (rate constant, 4.45 min-1). The results establish that the human E1alpha mutations in the putative thiamine pyrophosphate-binding pocket that are studied, with the exception of G204S-alpha, have no effect on E1 subunit assembly. The T265R-alpha mutation adversely impacts both E1alpha folding and subunit interactions. The mutations involving the C-terminal aromatic residues impede both the kinetics of subunit assembly and the formation of the native alpha2 beta2 structure.

3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗
PubMed1999

The activity of microsomal triglyceride transfer protein is essential for accumulation of triglyceride within microsomes in McA-RH7777 cells. A unified model for the assembly of very low density lipoproteins.

Previously, based on distinct requirement of microsomal triglyceride transfer protein (MTP) and kinetics of triglyceride (TG) utilization, we concluded that assembly of very low density lipoproteins (VLDL) containing B48 or B100 was achieved through different paths (Wang, Y. , McLeod, R. S., and Yao, Z. (1997) J. Biol. Chem. 272, 12272-12278). To test if the apparent dual mechanisms were accounted for by apolipoprotein B (apoB) length, we studied VLDL assembly using transfected cells expressing various apoB forms (e.g. B64, B72, B80, and B100). For each apoB, enlargement of lipoprotein to form VLDL via bulk TG incorporation was induced by exogenous oleate, which could be blocked by MTP inhibitor BMS-197636 treatment. While particle enlargement was readily demonstrable by density ultracentrifugation for B64- and B72-VLDL, it was not obvious for B80- and B100-VLDL unless the VLDL was further resolved by cumulative rate flotation into VLDL(1) (S(f) > 100) and VLDL(2) (S(f) 20-100). BMS-197636 diminished B100 secretion in a dose-dependent manner (0.05-0.5 microM) and also blocked the particle enlargement from small to large B100-lipoproteins. These results yield a unified model that can accommodate VLDL assembly with all apoB forms, which invalidates our previous conclusion. To gain a better understanding of the MTP action, we examined the effect of BMS-197636 on lipid and apoB synthesis during VLDL assembly. While BMS-197636 (0.2 microM) entirely abolished B100-VLDL(1) assembly/secretion, it did not affect B100 translation or translocation across the microsomal membrane, nor did it affect TG synthesis and cell TG mass. However, BMS-197636 drastically decreased accumulation of [(3)H]glycerol-labeled TG and TG mass within microsomal lumen. The decreased TG accumulation was not a result of impaired B100-VLDL assembly, because in cells treated with brefeldin A (0.2 microgram/ml), the assembly of B100-VLDL was blocked yet lumenal TG accumulation was normal. Thus, MTP plays a role in facilitating accumulation of TG within microsomes, a prerequisite for the post-translational assembly of TG-enriched VLDL.

Animals↗
PubMed2000

The assembly and secretion of apolipoprotein B-48-containing very low density lipoproteins in McA-RH7777 cells.

We have used an extraction procedure, which released membrane-bound apoB-100, to study the assembly of apoB-48 VLDL (very low density lipoproteins). This procedure released apoB-48, but not integral membrane proteins, from microsomes of McA-RH7777 cells. Upon gradient ultracentrifugation, the extracted apoB-48 migrated in the same position as the dense apoB-48-containing lipoprotein (apoB-48 HDL (high density lipoprotein)) secreted into the medium. Labeling studies with [(3)H]glycerol demonstrated that the HDL-like particle extracted from the microsomes contains both triglycerides and phosphatidylcholine. The estimated molar ratio between triglyceride and phosphatidylcholine was 0.70 +/- 0.09, supporting the possibility that the particle has a neutral lipid core. Pulse-chase experiments indicated that microsomal apoB-48 HDL can either be secreted as apoB-48 HDL or converted to apoB-48 VLDL. These results support the two-step model of VLDL assembly. To determine the size of apoB required to assemble HDL and VLDL, we produced apoB polypeptides of various lengths and followed their ability to assemble VLDL. Small amounts of apoB-40 were associated with VLDL, but most of the nascent chains associated with VLDL ranged from apoB-48 to apoB-100. Thus, efficient VLDL assembly requires apoB chains of at least apoB-48 size. Nascent polypeptides as small as apoB-20 were associated with particles in the HDL density range. Thus, the structural requirements of apoB to form HDL-like first-step particles differ from those to form second-step VLDL. Analysis of proteins in the d < 1.006 g/ml fraction after ultracentrifugation of the luminal content of the cells identified five chaperone proteins: binding protein, protein disulfide isomerase, calcium-binding protein 2, calreticulin, and glucose regulatory protein 94. Thus, intracellular VLDL is associated with a network of chaperones involved in protein folding. Pulse-chase and subcellular fractionation studies showed that apoB-48 VLDL did not accumulate in the rough endoplasmic reticulum. This finding indicates either that the two steps of apoB lipoprotein assembly occur in different compartment or that the assembled VLDL is transferred rapidly out of the rough endoplasmic reticulum.

Animals↗
PubMed2000

A human SCO2 mutation helps define the role of Sco1p in the cytochrome oxidase assembly pathway.

Deficiencies in cytochrome oxidase, the terminal enzyme of the mitochondrial respiratory chain, are most often caused by an inability to complete assembly of the enzyme. Pathogenic mutations in SCO2, which encodes a cytochrome oxidase assembly factor, were recently described in several cases of fatal infantile cardioencephalomyopathy. To determine the molecular etiology of these disorders, we describe the generation and characterization of the parallel mutations in the homologous yeast SCO1 gene. We show that the E155K yeast sco1 mutant is respiration-competent, whereas the S240F mutant is not. Interestingly, the S240F mutation allows partial but incorrect assembly of cytochrome oxidase, as judged by an altered cytochrome aa(3) peak. Immunoblot analysis reveals a specific absence of subunit 2 from the cytochrome oxidase in this mutant. Taken together, our data suggest that Sco1p provides copper to the Cu(A) site on subunit 2 at a step occurring late in the assembly pathway. This is the first instance of a yeast cytochrome oxidase assembly mutant that is partially assembled. The S240F mutant also represents a powerful new tool with which to elucidate further steps in the cytochrome oxidase assembly pathway.

Carrier Proteins↗
PubMed2000

Type IV collagen of the glomerular basement membrane. Evidence that the chain specificity of network assembly is encoded by the noncollagenous NC1 domains.

The ultrafiltration function of the glomerular basement membrane (GBM) of the kidney is impaired in genetic and acquired diseases that affect type IV collagen. The GBM is composed of five (alpha1 to alpha5) of the six chains of type IV collagen, organized into an alpha1.alpha2(IV) and an alpha3.alpha4.alpha5(IV) network. In Alport syndrome, mutations in any of the genes encoding the alpha3(IV), alpha4(IV), and alpha5(IV) chains cause the absence of the alpha3. alpha4.alpha5 network, which leads to progressive renal failure. In the present study, the molecular mechanism underlying the network defect was explored by further characterization of the chain organization and elucidation of the discriminatory interactions that govern network assembly. The existence of the two networks was further established by analysis of the hexameric complex of the noncollagenous (NC1) domains, and the alpha5 chain was shown to be linked to the alpha3 and alpha4 chains by interaction through their respective NC1 domains. The potential recognition function of the NC1 domains in network assembly was investigated by comparing the composition of native NC1 hexamers with hexamers that were dissociated and reconstituted in vitro and with hexamers assembled in vitro from purified alpha1-alpha5(IV) NC1 monomers. The results showed that NC1 monomers associate to form native-like hexamers characterized by two distinct populations, an alpha1.alpha2 and alpha3.alpha4.alpha5 heterohexamer. These findings indicate that the NC1 monomers contain recognition sequences for selection of chains and protomers that are sufficient to encode the assembly of the alpha1.alpha2 and alpha3.alpha4.alpha5 networks of GBM. Moreover, hexamer formation from the alpha3, alpha4, and alpha5 NC1 monomers required co-assembly of all three monomers, suggesting that mutations in the NC1 domain in Alport syndrome may disrupt the assembly of the alpha3.alpha4.alpha5 network by interfering with the assembly of the alpha3.alpha4.alpha5 NC1 hexamer.

Animals↗
PubMed2002

Altered amelogenin self-assembly based on mutations observed in human X-linked amelogenesis imperfecta (AIH1).

A hallmark of biological systems is a reliance on protein assemblies to perform complex functions. We have focused attention on mammalian enamel formation because it relies on a self-assembling protein complex to direct mineral habit. The principle protein of enamel is amelogenin, a 180-amino acid hydrophobic protein that self-assembles to form nanospheres. We have used independent technical methods, consisting of the yeast two-hybrid (Y2H) assay and surface plasmon resonance (SPR), to demonstrate the importance of amelogenin self-assembly domains. In addition, we have analyzed mutations in amelogenin observed in patients with amelogenesis imperfecta who demonstrate defects in enamel formation. Assessments of self-assembly of these mutant amelogenins by either SPR or Y2H assay yield concordant data. These data support the conclusion that the amelogenin amino-terminal self-assembly domain is essential to the creation of an enamel extracellular organic matrix capable of directing mineral formation. It also suggests that a pathway through which point mutations in the amelogenin protein can adversely impact on the formation of the enamel organ is by disturbing self-assembly of the organic matrix. These data support the utilization of the Y2H assay to search for protein interactions among extracellular matrix proteins that contribute to biomineralization and provide functional information on protein-protein and protein-mineral interactions.

Amelogenesis Imperfecta↗
PubMed2002

Two distinct domains of protein 4.1 critical for assembly of functional nuclei in vitro.

Protein 4.1R, a multifunctional structural protein, acts as an adaptor in mature red cell membrane skeletons linking spectrin-actin complexes to plasma membrane-associated proteins. In nucleated cells protein 4.1 is not associated exclusively with plasma membrane but is also detected at several important subcellular locations crucial for cell division. To identify 4.1 domains having critical functions in nuclear assembly, 4.1 domain peptides were added to Xenopus egg extract nuclear reconstitution reactions. Morphologically disorganized, replication deficient nuclei assembled when spectrin-actin-binding domain or NuMA-binding C-terminal domain peptides were present. However, control variant spectrin-actin-binding domain peptides incapable of binding actin or mutant C-terminal domain peptides with reduced NuMA binding had no deleterious effects on nuclear reconstitution. To test whether 4.1 is required for proper nuclear assembly, 4.1 isoforms were depleted with spectrin-actin binding or C-terminal domain-specific antibodies. Nuclei assembled in the depleted extracts were deranged. However, nuclear assembly could be rescued by the addition of recombinant 4.1R. Our data establish that protein 4.1 is essential for nuclear assembly and identify two distinct 4.1 domains, initially characterized in cytoskeletal interactions, that have crucial and versatile functions in nuclear assembly.

Actins↗
PubMed2003

Rpn5 is a conserved proteasome subunit and required for proper proteasome localization and assembly.

Proper function of the 26 S proteasome requires assembly of the regulatory complex, which is composed of the lid and base subcomplexes. We characterized Rpn5, a lid subunit, in fission yeast. We show that Rpn5 associates with the proteasome rpn5. Deletion (rpn5Delta) exacerbates the growth defects in proteasome mutants, leading to mitotic abnormalities, which correlate with accumulation of polyubiquitinated proteins, such as Cut2/securin. Rpn5 expression is tightly controlled; both overexpression and deletion of rpn5 impair proteasome functions. The proteasome is assembled around the inner nuclear membrane in wild-type cells; however, in rpn5Delta cells, proteasome subunits are improperly assembled and/or localized. In the lid mutants, Rpn5 is mislocalized in the cytosol, while in the base mutants, Rpn5 can enter the nucleus, but is left in the nucleoplasm, and not assembled into the nuclear membrane. These results suggest that Rpn5 is a dosage-dependent proteasome regulator and plays a role in mediating proper proteasome assembly. Moreover, the Rpn5 assembly may be a cooperative process that involves at least two steps: 1) nuclear import and 2) subsequent assembly into the nuclear membrane. The former step requires other components of the lid, while the latter requires the base. Human Rpn5 rescues the phenotypes associated with rpn5Delta and is incorporated into the yeast proteasome, suggesting that Rpn5 functions are highly conserved.

Carrier Proteins↗
PubMed2003

Identification and characterization of a common set of complex I assembly intermediates in mitochondria from patients with complex I deficiency.

Deficiencies in the activity of complex I (NADH: ubiquinone oxidoreductase) are an important cause of human mitochondrial disease. Complex I is composed of at least 46 structural subunits that are encoded in both nuclear and mitochondrial DNA. Enzyme deficiency can result from either impaired catalytic efficiency or an inability to assemble the holoenzyme complex; however, the assembly process remains poorly understood. We have used two-dimensional Blue-Native/SDS gel electrophoresis and a panel of 11 antibodies directed against structural subunits of the enzyme to investigate complex I assembly in the muscle mitochondria from four patients with complex I deficiency caused by either mitochondrial or nuclear gene defects. Immunoblot analyses of second dimension denaturing gels identified seven distinct complex I subcomplexes in the patients studied, five of which could also be detected in nondenaturing gels in the first dimension. Although the abundance of these intermediates varied among the different patients, a common constellation of subcomplexes was observed in all cases. A similar profile of subcomplexes was present in a human/mouse hybrid fibroblast cell line with a severe complex I deficiency due to an almost complete lack of assembly of the holoenzyme complex. The finding that diverse causes of complex I deficiency produce a similar pattern of complex I subcomplexes suggests that these are intermediates in the assembly of the holoenzyme complex. We propose a possible assembly pathway for the complex, which differs significantly from that proposed for Neurospora, the current model for complex I assembly.

Adult↗
PubMed2004

Assembly of exogenous fibronectin by fibronectin-null cells is dependent on the adhesive substrate.

The role of endogenously synthesized fibronectin (FN) in assembly was studied with cells lacking or expressing FN. Cells were cultured as homogeneous or mixed populations on surfaces coated with different matrix proteins. Compared with FN-expressing cells, FN-null cells poorly assembled exogenous plasma FN (pFN) when adhered to vitronectin or the recombinant cell-binding domain (III(7-10)) of FN. Vitronectin had a suppressive effect that was overcome by co-adsorbed pFN or laminin-1 but not by soluble FN. In co-cultures of FN-expressing cells and FN-null cells, endogenous FN was preferentially assembled around FN-expressing cells regardless of the adhesive ligand. If the adhesive ligand was vitronectin, exogenous pFN assembled preferentially around cells expressing cellular FN or recombinant EDa- or EDa+ FN. In co-cultures on vitronectin of FN-null cells and beta(1) integrin subunit-null cells, fibrils of cellular FN and pFN were preferentially deposited by FN-null (beta(1)-expressing) cells immediately adjacent to (FN-secreting) beta(1)-null cells. In co-cultures on vitronectin of FN-null cells and beta(1)-null cells expressing a chimera with the extracellular domain of beta(1) and the cytoplasmic domain of beta(3), preferential assembly was by the chimera-expressing cells. These results indicate that the adhesive ligand is a determinant of FN assembly by cells not secreting endogenous FN (suppressive if vitronectin, non-suppressive but non-supportive if III(7-10), supportive if pFN or laminin-1) and suggest that efficient interaction of freshly secreted cellular FN with a beta(1) integrin, presumably alpha(5)beta(1), substitutes for integrin-mediated adherence to a preformed matrix of laminin-1 or pFN to support assembly of FN.

Animals↗
PubMed2004

Gamma-secretase complex assembly within the early secretory pathway.

gamma-Secretase is an aspartyl protease complex composed of the four core components APH-1, nicastrin (NCT), presenilin (PS), and PEN-2. It catalyzes the final intramembranous cleavage of the beta-secretase-processed beta-amyloid precursor protein to liberate the neurotoxic amyloid beta-peptide. Whereas unassembled complex components appear to be unstable and/or to be retained within the endoplasmic reticulum (ER), the fully assembled complex is known to exert its biological function in late secretory compartments, including the plasma membrane. We thus hypothesized that the gamma-secretase complex undergoes a stepwise assembly within the ER. We demonstrate that gamma-secretase-associated NCT can be actively retained within the ER by the addition of a retention signal. Under these conditions, complex assembly occurred in the absence of maturation of NCT, and ER-retained immature NCT associated with APH-1, PEN-2, and PS fragments. Moreover, a biotinylated transition state gamma-secretase inhibitor allowed the preferential isolation of the fully assembled complex containing immature NCT. Furthermore, we observed a conformational change in immature NCT, which is known to be selectively associated with complete gamma-secretase complex assembly. This was also observed for a small amount of immature endogenous NCT. ER-retained NCT also rescued the biochemical phenotype observed upon RNA interference-mediated NCT knockdown, viz. reduced amyloid beta-peptide production; instability of PS, PEN-2, and APH-1; and accumulation of beta-amyloid precursor protein C-terminal fragments. Finally, we demonstrate that dimeric (NCT/APH-1) and trimeric (NCT/APH-1/PS) intermediates of gamma-secretase complex assembly containing endogenous NCT are retained within the ER and that the incorporation of the fourth and last binding partner (PEN-2) also occurs on immature NCT, suggesting a complete assembly of the gamma-secretase complex within the ER.

Amyloid Precursor Protein Secretases↗