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1.
OPA1 is a cause gene for autosomal dominant optic atrophy and possesses eight alternative splicing variants. Here, we identified two isoforms of OPA1 proteins in HeLa cells and examined their submitochondrial localization and complex formations. RT-PCR shows that HeLa cells mainly express isoforms 7 and 1 of OPA1. Since the third cleavage site is mainly utilized in HeLa cells, the predicted molecular masses of their processed proteins are consistent with the 93- and 88-kDa proteins. Biochemical examinations indicate that both of the OPA1 isoforms are present in the intermembrane space. Submitochondrial fractionation by sucrose density-gradient centrifugation shows that the 88-kDa protein predominantly associates with the mitochondrial outer membrane, on the contrary, the 93-kDa protein associates with the inner membrane. Gel filtration analysis indicates that they compose the different molecular mass complexes in mitochondria. These differences between two isoforms of OPA1 would suggest their crucial role involved in the mitochondrial membrane formation.  相似文献   

2.

Background

The S. cerevisiae α-factor receptor, Ste2p, is a G-protein coupled receptor that plays key roles in yeast signaling and mating. Oligomerization of Ste2p has previously been shown to be important for intracellular trafficking, receptor processing and endocytosis. However the role of ligand in receptor oligomerization remains enigmatic.

Methods

Using functional recombinant forms of purified Ste2p, atomic force microscopy, dynamic light scattering and chemical crosslinking are applied to investigate the role of ligand in Ste2p oligomerization.

Results

Atomic force microscopy images indicate a molecular height for recombinant Ste2p in the presence of α-factor nearly double that of Ste2p alone. This observation is supported by complementary dynamic light scattering measurements which indicate a ligand-induced increase in the polydispersity of the Ste2p hydrodynamic radius. Finally, chemical cross-linking of HEK293 plasma membranes presenting recombinant Ste2p indicates α-factor induced stabilization of the dimeric form and higher order oligomeric forms of the receptor upon SDS-PAGE analysis.

Conclusions

α-factor induces oligomerization of Ste2p in vitro and in membrane.

General significance

These results provide additional evidence of a possible role for ligand in mediation of Ste2p oligomerization in vivo.  相似文献   

3.
Swt1 is an RNA endonuclease that plays an important role in quality control of nuclear messenger ribonucleoprotein particles (mRNPs) in eukaryotes; however, its structural details remain to be elucidated. Here, we report the crystal structure of the C-terminal (CT) domain of Swt1 from Saccharomyces cerevisiae, which shares common characteristics of higher eukaryotes and prokaryotes nucleotide binding (HEPN) domain superfamily. To study in detail the full-length protein structure, we analyzed the low-resolution architecture of Swt1 in solution using small angle X-ray scattering (SAXS) method. Both the CT domain and middle domain exhibited a good fit upon superimposing onto the molecular envelope of Swt1. Our study provides the necessary structural information for detailed analysis of the functional role of Swt1, and its importance in the process of nuclear mRNP surveillance.  相似文献   

4.
5.
6.
Mitochondria are highly dynamic organelles that undergo frequent fusion and fission. The large GTPase optic atrophy 1 (OPA1) is identified as a core component of inner membrane (IM) fusion. OPA1 exists as the membrane-anchored L-OPA1 and the proteolytically cleavage soluble S-OPA1. Recently, we showed that OPA1 and mitochondria-localized lipid cardiolipin (CL) cooperate in heterotypic IM fusion [Ban et al., Nat. Cell Biol. 19 (2017) 856–863]. We reconstituted an in vitro membrane fusion reaction using purified human L-OPA1 and S-OPA1 expressed in silkworm and found that L-OPA1 on one side of the membrane and CL on the other side were sufficient for mitochondrial fusion. L-OPA1 is the major fusion-prone factor in heterotypic fusion. However, the role of S-OPA1 remains unknown as S-OPA1 promoted L-OPA1-dependent heterotypic membrane fusion and homotypic CL-containing membrane fusion, but S-OPA1 alone was not sufficient for heterotypic membrane fusion. L-OPA1- and CL-mediated heterotypic mitochondrial fusion was confirmed in living cells, but tafazzin (Taz1), the causal gene product of Barth syndrome, was not essential for mitochondrial fusion. Taz1-dependent CL maturation might have other roles in the remodeling of mitochondrial DNA nucleoids.  相似文献   

7.
We have developed a novel screening method that measures the kinetics and potencies of inhibitors of the yeast multidrug resistance pumps Pdr5p and Snq2p. The assay uses the potentiometric fluorescent probe diS-C3(3) (as a benchmark substrate of both pumps) to distinguish drugs with minimal effects on plasma membrane potential as a marker of side-effects on membrane function and integrity. Using FK506, its structural analog rapamycin and enniatin B, we showed that our assay can also be used to determine the minimum drug concentration causing an immediate inhibitory effect and to compare the inhibitory potencies of the drug on the two pumps. We found that the protonophore CCCP effectively inhibits the transport of diS-C3(3) by both pumps and confirmed the activation of membrane H+-ATPase by CCCP.  相似文献   

8.
Understanding the structural traits of subunit G is essential, as it is needed for V1VO assembly and function. Here solution NMR of the recombinant N- (G1-59) and C-terminal segment (G61-114) of subunit G, has been performed in the absence and presence of subunit d of the yeast V-ATPase. The data show that G does bind to subunit d via its N-terminal part, G1-59 only. The residues of G1-59 involved in d binding are Gly7 to Lys34. The structure of G1-59 has been solved, revealing an α-helix between residues 10 and 56, whereby the first nine- and the last three residues of G1-59 are flexible. The surface charge distribution of G1-59 reveals an amphiphilic character at the N-terminus due to positive and negative charge distribution at one side and a hydrophobic surface on the opposite side of the structure. The C-terminus exhibits a strip of negative residues. The data imply that G1-59-d assembly is accomplished by hydrophobic interactions and salt-bridges of the polar residues. Based on the recently determined NMR structure of segment E18-38 of subunit E of yeast V-ATPase and the presently solved structure of G1-59, both proteins have been docked and binding epitopes have been analyzed.  相似文献   

9.
10.
Yor1p, a Saccharomyces cerevisiae plasma membrane ABC-transporter, is associated to oligomycin resistance and to rhodamine B transport. Here, by using the overexpressing strain Superyor [A. Decottignies, A.M. Grant, J.W. Nichols, H. de Wet, D.B. McIntosh, A. Goffeau, ATPase and multidrug transport activities of the overexpressed yeast ABC protein Yor1p, J. Biol. Chem. 273 (1998) 12612-12622], we show that Yor1p also confers resistance to rhodamine 6G and to doxorubicin. In addition, Yor1p protects cells, although weakly, against tetracycline, verapamil, eosin Y and ethidium bromide. The basal ATPase activity of the overexpressed form of Yor1p was studied in membrane preparations. This activity is quenched upon addition of micromolar amounts of vanadate. Vmax and Km values of ∼ 0.8 s− 1 and 50 ± 8 μM are measured. Mutations of essential residues in the nucleotide binding domain 2 reduces the activity to that measured with a Δyor1 strain. ATP hydrolysis is strongly inhibited by the addition of potential substrates of the transporter. Covalent reaction of 8-azido-[α-32P]ATP with Yor1p is not sensitive to the presence of excess oligomycin. Thus, competition of the drug with ATP binding is unlikely. Finally, we inspect possible hypotheses accounting for substrate inhibition, rather than stimulation, of ATP hydrolysis by the membrane preparation.  相似文献   

11.
Mitochondria are highly dynamic organelles, and mitochondrial fission is a crucial step of apoptosis. Although Oma1 is believed to be responsible for long form Opa1 (L-Opa1) processing during mitochondrial fragmentation, whether and how Oma1 is involved in L-Opa1 processing and participates in the regulation of chemoresistance is unknown. Chemosensitive and chemoresistant ovarian (OVCA) and cervical (CECA) cancer cells were treated with cisplatin (CDDP). Mitochondrial dynamics and protein contents were assessed by immunofluorescence and Western blot, respectively. The requirements of Oma1 and p53 for CDDP-induced L-Opa1 processing, mitochondrial fragmentation, and apoptosis were examined by siRNA or cDNA. CDDP induces L-Opa1 processing and mitochondrial fragmentation in chemosensitive but not in chemoresistant cells. CDDP induced Oma1 40-kDa form increases in OV2008 cells, not in C13* cells. Oma1 knockdown inhibited L-Opa1 processing, mitochondrial fragmentation, and apoptosis. Silencing p53 expression attenuated the effects of CDDP in Oma1 (40 kDa) increase, L-Opa1 processing, mitochondrial fragmentation, and apoptosis in chemosensitive OVCA cells, whereas reconstitution of p53 in p53 mutant or null chemoresistant OVCA cells induced Oma1 (40 kDa) increase, L-Opa1 processing, mitochondrial fragmentation, and apoptosis irrespective of the presence of CDDP. Prohibitin 1 (Phb1) dissociates from Opa1-Phb1 complex and binds phosphorylated p53 (serine 15) in response to CDDP in chemosensitive but not chemoresistant CECA cells. These findings demonstrate that (a) p53 and Oma1 mediate L-Opa1 processing, (b) mitochondrial fragmentation is involved in CDDP-induced apoptosis in OVCA and CECA cells, and (c) dysregulated mitochondrial dynamics may in part be involved in the pathophysiology of CDDP resistance.  相似文献   

12.
Constitutively expressed HABP1 in normal murine fibroblast cell line induces growth perturbation, morphological abnormalities along with initiation of apoptosis. Here, we demonstrate that though HABP1 accumulation started in mitochondria from 48 hr of growth, induction of apoptosis with the release of cytochrome c and apoptosome complex formation occurred only after 60 hr. This mitochondrial dysfunction was due to gradual increase in ROS generation in HABP1 overexpressing cells. Along with ROS generation, increased Ca 2+ influx in mitochondria leading to drop in membrane potential was evident. Interestingly, upon expression of HABP1, the respiratory chain complex I was shown to be significantly inhibited. Electronmicrograph confirmed defective mitochondrial ultrastructure. The reduction in oxidant generation and drop in apoptotic cell population accomplished by disruption of HABP1 expression, corroborating the fact that excess ROS generation in HABP1 overexpressing cells leading to apoptosis was due to mitochondrial HABP1 accumulation.  相似文献   

13.
Arc35p, a component of the Arp2/3 complex, plays at least two distinct roles, regulating the actin cytoskeleton, but also microtubule function during cell division. Both functions involve calmodulin (CMD1). To investigate the pathway affecting microtubule function, we identified genes that are able to suppress the temperature-sensitive growth defect of the arc35-1 strain. Genes encoding gamma-tubulin (TUB4) or any subunit of casein kinase II (CKII) suppressed this growth defect, but did not suppress the growth defect of a mutant in another subunit of the Arp2/3 complex, arp2-1. We could also show a physical association of Arc35p with subunits of CKII, Cmd1p, and Tub4p. Based on the exclusive localization of Arc35p to the cytosolic Arp2/3 complex and on mutant phenotypes, we propose that the role of the Arc35p/CKII interaction might be to activate a cytosolic pool of gamma-tubulin, likely via calmodulin, for its nuclear and/or cytoplasmic functions.  相似文献   

14.
15.
Mitochondrial dysfunction is involved in the underlying pathology of Parkinson’s Disease (PD). PINK1 deficiency, which gives rise to familial early-onset PD, is associated with this dysfunction as well as increased oxidative stress. We have established primary fibroblast cell lines from two patients with PD who carry mutations in the PINK1 gene. The phosphorylation of Akt is abrogated in the presence of oxidative stressors in the complete absence of PINK1 suggesting enhanced apoptotic signalling. We have found an imbalance between the production of reactive oxygen species where the capacity of the cell to remove these toxins by anti-oxidative enzymes is greatly reduced. The expression levels of the anti-oxidant enzymes glutathione peroxidase-1, MnSOD, peroxiredoxin-3 and thioredoxin-2 were diminished. The p66Shc adaptor protein has recently been identified to become activated by oxidative stress by phosphorylation at residue Ser36 which then translocates to the mitochondrial inner membrane space. The phosphorylation of p66Shc at Ser36 is significantly increased in PINK1 deficient cell lines under normal tissue culture conditions, further still in the presence of compounds which elicit oxidative stress. The stable transfection of PINK1 in the fibroblasts which display the null phenotype ameliorates the hyper-phosphorylation of p66Shc.  相似文献   

16.
目的:构建p21WAF1/CIP1基因小干扰RNA(siRNA)的真核表达载体,观察其对p21WAF1/CIP1表达的影响和细胞周期的变化。方法:合成了针对p21WAF1/CIP1基因的siRNA,将其克隆到siRNA表达载体pSliencer2.1-U6neo上,将重组质粒和带FLAG标签的p21WAF1/CIP1共转染293T人胚肾细胞,通过Westernblot检验RNA干扰(RNAi)敲低外源p21WAF1/CIP1的效果;将重组质粒单独转染293T人胚肾细胞,利用p21WAF1/CIP1抗体检测RNAi敲低内源p21WAF1/CIP1的效果;利用流式细胞仪检测敲低后细胞周期的变化。结果:测序证明构建了p21WAF1/CIP1siRNA真核表达载体;Westernblot和流式细胞分析证明,构建的siRNA能有效降低p21WAF1/CIP1基因的表达,并且使G1期细胞数减少14.03%,S期细胞增多13.45%。结论:构建了p21WAF1/CIP1siRNA的真核表达载体,该siRNA能有效抑制p21WAF1/CIP1基因的表达并部分解除了G1期阻滞。  相似文献   

17.
Phosphatidylserine decarboxylase 1 (Psd1p) catalyzes the formation of the majority of phosphatidylethanolamine (PE) in the yeast Saccharomyces cerevisiae. Psd1p is localized to mitochondria, anchored to the inner mitochondrial membrane (IMM) through membrane spanning domains and oriented towards the mitochondrial intermembrane space. We found that Psd1p harbors at least two inner membrane-associated domains, which we named IM1 and IM2. IM1 is important for proper orientation of Psd1p within the IMM (Horvath et al., J. Biol. Chem. 287 (2012) 36744–55), whereas it remained unclear whether IM2 is important for membrane-association of Psd1p. To discover the role of IM2 in Psd1p import, processing and assembly into the mitochondria, we constructed Psd1p variants with deletions in IM2. Removal of the complete IM2 led to an altered topology of the protein with the soluble domain exposed to the matrix and to decreased enzyme activity. Psd1p variants lacking portions of the N-terminal moiety of IM2 were inserted into IMM with an altered topology. Psd1p variants with deletions of C-terminal portions of IM2 accumulated at the outer mitochondrial membrane and lost their enzyme activity. In conclusion we showed that IM2 is essential for full enzymatic activity, maturation and correct integration of yeast Psd1p into the inner mitochondrial membrane.  相似文献   

18.
YouJin Lee  Conrad C. Weihl 《Autophagy》2017,13(9):1615-1616
Macroautophagy/autophagy can be a selective degradative process via the utilization of various autophagic receptor proteins. Autophagic receptors selectively recognize ubiquitinated cargoes and deliver them to phagophores, the precursors to autophagosomes, for their degradation. For example, SQSTM1/p62 directly binds to ubiquitinated protein aggregates via its UBA domain and sequesters them into inclusion bodies via its PB1 domain. SQSTM1also interacts with phagophores via its LC3-interacting (LIR) motif. However, a regulatory mechanism for autophagic receptors is not yet understood.  相似文献   

19.
Proliferating germ cells in Caenorhabditiselegans provide a useful model system for deciphering fundamental mechanisms underlying the balance between proliferation and differentiation. Using gene expression profiling, we identified approximately 200 genes upregulated in the proliferating germ cells of C. elegans. Functional characterization using RNA-mediated interference demonstrated that over forty of these factors are required for normal germline proliferation and development. Detailed analysis of two of these factors defined an important regulatory relationship controlling germ cell proliferation. We established that the kinase VRK-1 is required for normal germ cell proliferation, and that it acts in part to regulate CEP-1(p53) activity. Loss of cep-1 significantly rescued the proliferation defects of vrk-1 mutants. We suggest that VRK-1 prevents CEP-1 from triggering an inappropriate cell cycle arrest, thereby promoting germ cell proliferation. This finding reveals a previously unsuspected mechanism for negative regulation of p53 activity in germ cells to control proliferation.  相似文献   

20.

Background

The enzymatic activity of the four proteases found in the house dust mite Dermatophagoides pteronyssinus is involved in the pathogenesis of allergy. Our aim was to elucidate the activation cascade of their corresponding precursor forms and particularly to highlight the interconnection between proteases during this cascade.

Methods

The cleavage of the four peptides corresponding to the mite zymogen activation sites was studied on the basis of the Förster Resonance Energy Transfer method. The proDer p 6 zymogen was then produced in Pichia pastoris to elucidate its activation mechanism by mite proteases, especially Der p 1. The role of the propeptide in the inhibition of the enzymatic activity of Der p 6 was also examined. Finally, the Der p 1 and Der p 6 proteases were localised via immunolocalisation in D. pteronyssinus.

Results

All peptides were specifically cleaved by Der p 1, such as proDer p 6. The propeptide of proDer p 6 inhibited the proteolytic activity of Der p 6, but once cleaved, it was degraded by the protease. The Der p 1 and Der p 6 proteases were both localised to the midgut of the mite.

Conclusions

Der p 1 in either its recombinant form or in the natural context of house dust mite extracts specifically cleaves all zymogens, thus establishing its role as a major activator of both mite cysteine and serine proteases.

General significance

This finding suggests that Der p 1 may be valuable target against mites.  相似文献   

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