共查询到20条相似文献,搜索用时 15 毫秒
1.
Identification of a 349-kilodalton protein (Gli349) responsible for cytadherence and glass binding during gliding of Mycoplasma mobile
下载免费PDF全文
![点击此处可从《Journal of bacteriology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Several mycoplasma species are known to glide in the direction of the membrane protrusion (head-like structure), but the mechanism underlying this movement is entirely unknown. To identify proteins involved in the gliding mechanism, protein fractions of Mycoplasma mobile were analyzed for 10 gliding mutants isolated previously. One large protein (Gli349) was observed to be missing in a mutant m13 deficient in hemadsorption and glass binding. The predicted amino acid sequence indicated a 348,758-Da protein that was truncated at amino acid residue 1257 in the mutant. Immunofluorescence microscopy with a monoclonal antibody showed that Gli349 is localized at the head-like protrusion's base, which we designated the cell neck, and immunoelectron microscopy established that the Gli349 molecules are distributed all around this neck. The number of Gli349 molecules on a cell was estimated by immunoblot analysis to be 450 +/- 200. The antibody inhibited both the hemadsorption and glass binding of M. mobile. When the antibody was used to treat gliding mycoplasmas, the gliding speed and the extent of glass binding were inhibited to similar extents depending on the concentration of the antibody. This suggested that the Gli349 molecule is involved not only in glass binding for gliding but also in movement. To explain the present results, a model for the mechanical cycle of gliding is discussed. 相似文献
2.
Identification of a 521-kilodalton protein (Gli521) involved in force generation or force transmission for Mycoplasma mobile gliding
下载免费PDF全文
![点击此处可从《Journal of bacteriology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Several mycoplasma species are known to glide on solid surfaces such as glass in the direction of the membrane protrusion, but the mechanism underlying this movement is unknown. To identify a novel protein involved in gliding, we raised monoclonal antibodies against a detergent-insoluble protein fraction of Mycoplasma mobile, the fastest glider, and screened the antibodies for inhibitory effects on gliding. Five monoclonal antibodies stopped the movement of gliding mycoplasmas, keeping them on the glass surface, and all of them recognized a large protein in immunoblotting. This protein, named Gli521, is composed of 4,738 amino acids, has a predicted molecular mass of 520,559 Da, and is coded downstream of a gene for another gliding protein, Gli349, which is known to be responsible for glass binding during gliding. Edman degradation analysis indicated that the N-terminal region is processed at the peptide bond between the amino acid residues at positions 43 and 44. Analysis of gliding mutants isolated previously revealed that the Gli521 protein is missing in a nonbinding mutant, m9, where the gli521 gene is truncated by a nonsense mutation at the codon for the amino acid at position 1170. Immunofluorescence and immunoelectron microscopy indicated that Gli521 localizes all around the base of the membrane protrusion, at the "neck," as previously observed for Gli349. Analysis of the inhibitory effects of the anti-Gli521 antibody on gliding motility revealed that this protein is responsible for force generation or force transmission, a role distinct from that of Gli349, and also suggested conformational changes of Gli349 and Gli521 during gliding. 相似文献
3.
A protein with a molecular mass of 42 kDa (P42) from Mycoplasma mobile, one of several mycoplasmas that exhibit gliding motility, was shown to be a novel NTPase (nucleoside triphosphatase). Although the P42 protein lacks a common ATP-binding sequence motif (Walker A), the recombinant proteins expressed in Escherichia coli certainly hydrolysed some nucleoside triphosphates, including ATP. The results of photoaffinity labelling by an ATP analogue supported that the P42 protein contains a specific binding site for ATP (or another nucleoside triphosphate). In the M. mobile genome, the P42 gene is located downstream of gli123, gli349 and gli521 genes, and they have been reported to be polycis-tronically transcribed. As the huge proteins encoded by gli123, gli349 and gli521 play a role in gliding motility of M. mobile, P42 might also have some kind of function in the gliding motility. The gliding motility of M. mobile is driven directly by ATP hydrolysis, but the key ATPase has not been identified. Our results showed that, among these four proteins, only P42 exhibited ATPase activity. Biochemical characteristics--optimal conditions for activity, substrate specificities, and inhibiting effects by ATP analogues--of the recombinant P42 proteins were very similar to those of a putative ATPase speculated from a previous analysis with a gliding 'ghost' whose cell membrane was permeabilized by Triton X-100. These results support the hypothesis that the P42 protein is the key ATPase in the gliding motility of M. mobile. 相似文献
4.
Morphology of isolated Gli349, a leg protein responsible for Mycoplasma mobile gliding via glass binding, revealed by rotary shadowing electron microscopy
下载免费PDF全文
![点击此处可从《Journal of bacteriology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Several species of mycoplasmas rely on an unknown mechanism to glide across solid surfaces in the direction of a membrane protrusion at the cell pole. Our recent studies on the fastest species, Mycoplasma mobile, suggested that a 349-kDa protein, Gli349, localized at the base of the membrane protrusion called the neck, forms legs that stick out from the neck and propel the cell by repeatedly binding to and releasing from a solid surface, based on the energy of ATP hydrolysis. Here, the Gli349 protein was isolated from mycoplasma cells and its structure was analyzed. Gel filtration analysis showed that the isolated Gli349 protein is monomeric. Rotary shadowing electron microscopy revealed that the molecular structure resembles the symbol for an eighth note in music. It contains an oval foot 14 nm long in axis. From this foot extend three rods in tandem of 43, 20, and 20 nm, in that order. The hinge connecting the first and second rods is flexible, while the next hinge has a distinct preference in its angle, near 90 degrees. Molecular images revealed that a monoclonal antibody that can bind to the position at one-third of the total peptide length from the N terminus bound to a position two-thirds from the foot end, suggesting that the foot corresponds to the C-terminal region. The amino acid sequence was assigned to the molecular image, and the topology of the molecule in the gliding machinery is discussed. 相似文献
5.
Molecular shape and binding force of Mycoplasma mobile’s leg protein Gli349 revealed by an AFM study
Charles Lesoil Hiroshi Sekiguchi Makoto Miyata Atsushi Ikai 《Biochemical and biophysical research communications》2010,391(3):1312-572
Recent studies of the gliding bacteria Mycoplasma mobile have identified a family of proteins called the Gli family which was considered to be involved in this novel and yet fairly unknown motility system. The 349 kDa protein called Gli349 was successfully isolated and purified from the bacteria, and electron microscopy imaging and antibody experiments led to the hypothesis that it acts as the “leg” of M. mobile, responsible for attachment to the substrate as well as for gliding motility. However, more precise evidence of the molecular shape and function of this protein was required to asses this theory any further. In this study, an atomic force microscope (AFM) was used both as an imaging and a force measurement device to provide new information about Gli349 and its role in gliding motility. AFM images of the protein were obtained revealing a complex structure with both rigid and flexible parts, consistent with previous electron micrographs of the protein. Single-molecular force spectroscopy experiments were also performed, revealing that Gli349 is able to specifically bind to sialyllactose molecules and withstand unbinding forces around 70 pN. These findings strongly support the idea that Gli349 is the “leg” protein of M. mobile, responsible for binding and also most probably force generation during gliding motility. 相似文献
6.
M Zushi K Gomi G Honda S Kondo S Yamamoto T Hayashi K Suzuki 《The Journal of biological chemistry》1991,266(30):19886-19889
The last three consecutive epidermal growth factor (EGF)-like structures of human thrombomodulin constitute the functional domain for protein C-activating cofactor activity and anticoagulant activity. Using site-directed deletion mutagenesis, we found that amino acid Asp349 of TME456, a recombinantly produced protein consisting of EGF-like structures 4, 5, and 6, is essential for retaining full protein C-activating cofactor activity. To investigate the role of Asp349 in the protein C-activating cofactor activity of human thrombomodulin, we have constructed two mutants of TMD123, a recombinantly produced protein consisting of domains D1, D2, and D3 of thrombomodulin, using site-directed point mutagenesis of the thrombomodulin coding sequence. In mutant TMD123A, the Asp349 codon was replaced with an Ala codon and in mutant TMD123E, the Asp349 codon was replaced with a Glu codon. The partially purified mutant proteins were assayed for their protein C-activating cofactor activity at various Ca2+ concentrations. TMD123 and TMD123E protein showed similar high levels of cofactor activity and similar patterns of Ca2+ dependence, while TMD123A had lower cofactor activity and did not show any Ca2+ dependence. We concluded that Asp349 in the fourth EGF-like structure of human thrombomodulin plays a role in its Ca(2+)-mediated binding to protein C. 相似文献
7.
Mycoplasma mobile binds to solid surfaces and glides smoothly and continuously by a unique mechanism. A huge protein, Gli521 (521 kDa), is involved in the gliding machinery, and it is localized in the cell neck, the base of the membrane protrusion. This protein is thought to have the role of force transmission. In this study, the Gli521 protein was purified from M. mobile cells, and its molecular shape was studied. Gel filtration analysis showed that the isolated Gli521 protein forms mainly a monomer in Tween 80-containing buffer and oligomers in Triton X-100-containing buffer. Rotary shadowing electron microscopy showed that the Gli521 monomer consisted of three parts: an oval, a rod, and a hook. The oval was 15 nm long by 11 nm wide, and the filamentous part composed of the rod and the hook was 106 nm long and 3 nm in diameter. The Gli521 molecules form a trimer, producing a “triskelion” reminiscent of eukaryotic clathrin, through association at the hook end. Image averaging of the central part of the triskelion suggested that there are stable and rigid structures. The binding site of a previously isolated monoclonal antibody on Gli521 images showed that the hook end and oval correspond to the C- and N-terminal regions, respectively. Partial digestion of Gli521 showed that the molecule could be divided into three domains, which we assigned to the oval, rod, and hook of the molecular image. The Gli521 molecule''s role in the gliding mechanism is discussed.Mycoplasmas are commensal and occasionally parasitic bacteria with small genomes that lack a peptidoglycan layer (31). Several mycoplasma species form membrane protrusions, such as the headlike structure in Mycoplasma mobile and the attachment organelle in Mycoplasma pneumoniae (15, 19, 21, 22, 25, 33, 34, 36). On solid surfaces, these species exhibit gliding motility in the direction of the protrusion; this motility is believed to be involved in the pathogenicity of mycoplasmas (12, 13, 16, 20, 21). Interestingly, mycoplasmas have no surface flagella or pili, and their genomes contain no genes related to other known bacterial motility systems. In addition, no homologs of motor proteins that are common in eukaryotic motility have been found (11).M. mobile, which was isolated from the gills of a freshwater fish in the early 1980s, is a fast gliding mycoplasma (14). It glides smoothly and continuously on glass at an average speed of 2.0 to 4.5 μm/s, or three to seven times the length of the cell per second, exerting a force of up to 27 pN (8, 9, 24, 25, 32). Previously, we identified huge proteins involved in this gliding mechanism that are localized at the so-called cell neck, the base of the membrane protrusion (17, 26, 30, 35, 37, 39); we also visualized the putative machinery and the binding protein (1, 18, 23) and identified both the direct energy source used and the direct binding target (10, 27, 38). The force generated by the gliding machinery may be supported from inside the cell by a cytoskeletal “jellyfish” structure (28, 29). On the basis of these results, we proposed a working model, called the centipede or power stroke model, where cells are propelled by “legs” composed of Gli349 that repeatedly catch and release sialic acids fixed on the glass surface (5, 19, 21). These legs are driven by the force exerted by P42 through Gli521 molecules, which is supported by the jellyfish structure, based on energy from ATP hydrolysis.The Gli521 protein, which has an unusually high molecular mass (521 kDa), is suggested to have the role of force transmission, because a monoclonal antibody against this protein stops gliding, keeping the cells on a solid surface (35). About 450 molecules are estimated to be clustered in the gliding machinery with other component proteins, although their alignment has not been clarified (35, 37, 39). In this study, we isolated the Gli521 protein and studied its molecular shape using electron microscopy (EM) and biochemical analyses in order to understand the gliding mechanism. 相似文献
8.
The nucleotide sequence of a Sau3A1 restriction nuclease fragment that complemented an Escherichia coli chlD::Mu cts mutant strain was determined. DNA and deduced amino acid sequence analysis revealed two open reading frames (ORFs) that potentially codes for proteins with amino acid sequence homology with binding protein-dependent transport systems. One of the ORFs showed a sequence that encoded a protein with properties that were characteristic of a hydrophobic inner membrane protein. The other ORF, which was responsible for complementing a chlD mutant, encoded a protein with conserved sequences in nucleotide-binding proteins and hydrophilic inner membrane proteins in active transport systems. A proposal that the chlD locus is the molybdate transport operon is discussed in terms of the chlD phenotype. 相似文献
9.
Calcitonin receptor-like receptor (CRLR) constitutes either a CGRP receptor when complexed with receptor activity-modifying protein 1 (RAMP1) or an adrenomedullin receptor when complexed with RAMP2 or RAMP3. RAMP proteins modify the glycosylation status of CRLR and determine their receptor specificity; when treated with tunicamycin, a glycosylation inhibitor, CHO-K1 cells constitutively expressing both RAMP2 and CRLR lost the capacity to bind adrenomedullin. Similarly, in HEK293 EBNA cells constitutively expressing RAMP1/CRLR receptor complex CGRP binding was remarkably inhibited. Whichever RAMP protein was co-expressing with CRLR, the ligand binding was sensitive to tunicamycin. There are three putative Asn-linked glycosylation sites in the extracellular, amino terminal domain of CRLR at positions 66, 118 and 123. Analysis of CRLR mutants in which Gln was substituted for selected Asn residues showed that glycosylation of Asn123 is required for both the binding of adrenomedullin and the transduction of its signal. Substituting Asn66 or Asn118 had no effect. FACS analysis of cells expressing FLAG-tagged CRLRs showed that disrupting Asn-linked glycosylation severely affected the transport of the CRLR protein to the cell surface on N66/118/123Q mutant, and slightly reduced the level of the cell surface expression of N123Q mutant compared with wild-type CRLR. But other single mutants (N66Q, N118Q) had no effect for other single mutants. Our data shows that glycosylation of Asn66 and Asn118 is not essential for ligand binding, signal transduction and cell surface expression, and Asn123 is important for ligand binding and signal transduction rather than cell surface expression. It thus appears that glycosylation of Asn123 is required for CRLR to assume the appropriate conformation on the cell surface through its interaction with RAMPs. 相似文献
10.
11.
Adam Liwo Kenneth D. Gibson Harold A. Scheraga Paul W. Brandt-Rauf Regina Monaco Matthew R. Pincus 《Journal of Protein Chemistry》1994,13(2):237-251
Oncogenic p21 protein, encoded by theras-oncogene, that causes malignant transformation of normal cells and many human tumors, is almost identical in sequence to its normal protooncogene-encoded counterpart protein, except for the substitution of arbitrary amino acids for the normally occurring amino acids at critical positions such as Gly 12 and Gin 61. Since p21 is normally activated by the binding of GTP in place of GDP, it has been postulated that oncogenic forms must retain bound GTP for prolonged time periods. However, two multiply substituted p21 proteins have been cloned, neither of which binds GDP or GTP. One of these mutant proteins with Val for Gly 10, Arg for Gly 12, and Thr for Ala 59 causes cell transformation, while the other, similar protein with Gly 10, Arg 12, Val for Gly 13 and Thr 59 does not transform cells. To define the critical conformational changes that occur in the p21 protein that cause it to become oncogenic, we have calculated the low energy conformations of the two multiply substituted mutant p21 proteins using a new adaptation of the electrostatically driven Monte Carlo (EDMC) technique, based on the program ECEPP. We have used this method to explore the conformational space available to both proteins and to compute the average structures for both using statistical mechanical averaging. Comparison of the average structures allows us to detect the major differences in conformation between the two proteins. Starting structures for each protein were calculated using the recently deposited x-ray crystal coordinates for the p21 protein, that was energy-refined using ECEPP, and then perturbed using the EDMC method to compute its average structure. The specific amino acid substitutions for both proteins were then generated into the lowest energy structure generated by this procedure, subjected to energy minimization and then to full EDMC perturbations. We find that both mutant proteins exhibit major differences in conformation in specific regions, viz., residues 35–47, 55–78, 81–93, 96–110, 115–126, and 123–134, compared with the EDMC-refined x-ray structure of the wild-type protein. These regions have been found to be the most flexible in the p21 protein bound to GDP from prior molecular dynamics calculations (Dykeset al., 1993). Comparison of the EDMC-average structure of the transforming mutant with that of the nontransforming mutant reveals major structural differences at residues 10–16, 32–40, and 60–68. These structural differences appear to be the ones that are critical in activation of the p21 protein. Analysis of the correlated motions of the different regions of the two mutant proteins reveals that changes in the conformation of regions in the carboxyl half of the protein are caused by changes in conformation around residues 10–16 and are transmitted by means of residues around Gln 61. The latter region therefore constitutes a molecular switch unit, in agreement with conclusions from prior work.On leave from the Department of Chemistry, University of Gdask, ul. Sobieskiego 18, 80-952 Gdask, Poland. 相似文献
12.
Intersection of the Kap123p-mediated nuclear import and ribosome export pathways 总被引:5,自引:0,他引:5
下载免费PDF全文
![点击此处可从《Molecular and cellular biology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Sydorskyy Y Dilworth DJ Yi EC Goodlett DR Wozniak RW Aitchison JD 《Molecular and cellular biology》2003,23(6):2042-2054
Kap123p is a yeast beta-karyopherin that imports ribosomal proteins into the nucleus prior to their assembly into preribosomal particles. Surprisingly, Kap123p is not essential for growth, under normal conditions. To further explore the role of Kap123p in nucleocytoplasmic transport and ribosome biogenesis, we performed a synthetic fitness screen designed to identify genes that interact with KAP123. Through this analysis we have identified three other karyopherins, Pse1p/Kap121p, Sxm1p/Kap108p, and Nmd5p/Kap119p. We propose that, in the absence of Kap123p, these karyopherins are able to supplant Kap123p's role in import. In addition to the karyopherins, we identified Rai1p, a protein previously implicated in rRNA processing. Rai1p is also not essential, but deletion of the RAI1 gene is deleterious to cell growth and causes defects in rRNA processing, which leads to an imbalance of the 60S/40S ratio and the accumulation of halfmers, 40S subunits assembled on polysomes that are unable to form functional ribosomes. Rai1p localizes predominantly to the nucleus, where it physically interacts with Rat1p and pre-60S ribosomal subunits. Analysis of the rai1/kap123 double mutant strain suggests that the observed genetic interaction results from an inability to efficiently export pre-60S subunits from the nucleus, which arises from a combination of compromised Kap123p-mediated nuclear import of the essential 60S ribosomal subunit export factor, Nmd3p, and a DeltaRAI1-induced decrease in the overall biogenesis efficiency. 相似文献
13.
Gliding motility of Mycoplasma mobile can occur by repeated binding to N-acetylneuraminyllactose (sialyllactose) fixed on solid surfaces
下载免费PDF全文
![点击此处可从《Journal of bacteriology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Mycoplasma mobile relies on an unknown mechanism to glide across solid surfaces including glass, animal cells, and plastics. To identify the direct binding target, we examined the factors that affect the binding of Mycoplasma pneumoniae to solid surfaces and concluded that N-acetylneuraminyllactose (sialyllactose) attached to a protein can mediate glass binding on the basis of the following four lines of evidence: (i) glass binding was inhibited by N-acetylneuraminidase, (ii) glass binding was inhibited by N-acetylneuraminyllactose in a structure-dependent manner, (iii) binding occurred on glass pretreated with bovine serum albumin attached to N-acetylneuraminyllactose, and (iv) gliding speed depended on the density of N-acetylneuraminyllactose on glass. 相似文献
14.
Membrane cofactor protein (MCP) of the C system is a widely distributed regulatory molecule with C3b/C4b binding and factor I-dependent cofactor activity. A rabbit polyclonal antibody was raised against purified human MCP, and it was found to also immunoprecipitate C4b-binding protein (C4bp). Other related complement regulatory proteins, factor H, C3b/C4b receptor, and decay-accelerating factor, were not recognized by this polyclonal antibody to MCP. The cross-reactive epitope was sensitive to reduction with 2-ME and about 3% of the anti-MCP antibody reacted with C4bp. The amino-terminal 48,000-Da, chymotryptic fragment of C4bp was recognized by the antibody to MCP. This fragment of C4bp contains a seven-amino acid peptide that is identical, in its sequence and its location in the third short consensus repeat, to one found in MCP. Two polyclonal antibodies to C4bp, one raised to native and the other to reduced C4bp, did not cross-react with MCP. In addition to this one-way cross-reaction with C4bp, a protein with a m.w. of approximately 60,000 (p60) was found in two of three C4bp preparations that also cross-reacted with antiserum to MCP. p60 was present in trace quantities in the C4bp preparation and was successfully isolated from plasma by C3b affinity chromatography. Its Mr was distinct from that of MCP and other known C3b/C4b binding proteins. Furthermore, p60 was isolated by two different procedures and such material possessed no detectable cofactor activity. Based on these results, p60 is a plasma C3b-binding protein that shares epitopes with C4bp and MCP, and is probably not a soluble form of MCP. 相似文献
15.
Yahiro K Niidome T Kimura M Hatakeyama T Aoyagi H Kurazono H Imagawa Ki Wada A Moss J Hirayama T 《The Journal of biological chemistry》1999,274(51):36693-36699
Helicobacter pylori, a Gram-negative gastric bacterium, secretes VacA, a cytotoxin that causes vacuolar degeneration of susceptible cells. Velocity sedimentation analysis showed that treatment of VacA at alkaline pH led to disassembly of VacA oligomers, an observation reported previously for acid-treated VacA. Exposure of VacA to acid or alkali increased its binding to AZ-521 cells, as shown by indirect immunofluorescence and flow cytometry. Moreover, immunoprecipitates with polyclonal antibodies against VacA from AZ-521 cells previously exposed to acid- or alkali-treated VacA had a 250-kDa glycoprotein containing galactose-beta(1-3)-N-acetylgalactosamine and galactose-beta(1-4)-N-acetylglucosamine. p250, purified by chromatography on peanut agglutinin affinity and Superose 6 columns, contained N-terminal and internal amino acid sequences of YRQQRKLVEEIGWSYT and LIIQDHILEATQDDY, respectively. These sequences are identical to those of a receptor protein-tyrosine phosphatase (RPTPbeta/PTPzeta); in agreement, p250 reacted with anti-human RPTPbeta monoclonal antibody. Immunoprecipitation with anti-human RPTPbeta antibody of solubilized membrane preparations previously incubated with VacA or heat-inactivated VacA demonstrated that RPTPbeta bound native, but not denatured, VacA. Acidic and alkaline treatments were associated with activation of VacA and increased binding to the cell surface RPTPbeta. 相似文献
16.
【目的】制备人细小病毒B19-VP1u的多克隆抗体,探究VP1u多克隆抗体及其保守区外N端氨基酸对病毒磷脂酶A2活性的影响。【方法】首先通过分子克隆方法构建相应原核表达载体;利用原核表达系统纯化含MBP标签的VP1u全长及N端系列截短突变融合蛋白;接着免疫新西兰大白兔制备全长VP1u蛋白的多克隆抗体;最后利用磷脂酶A2活性检测试剂盒检测了纯化蛋白的磷脂酶A2活性。【结果】Western blot及免疫荧光实验证实制备的多克隆抗体具有较高的特异性;磷脂酶A2活性检测发现全长VP1u-MBP融合蛋白具有一定的活性,该活性可以被VP1u的抗体抑制;N端保守区外截短系列蛋白的酶活检测发现,N端截掉12个氨基酸时酶活降低53%,截掉67个氨基酸时酶活性几乎完全丧失。【结论】首次发现VP1u保守区外N端氨基酸,尤其是第12个氨基酸前的区域以及第22-67个氨基酸之间的区域,对sPLA2活性的保持具有重要意义,推测该区域可能对维持正常的蛋白构象起重要的作用;而其特异性多克隆抗体的制备也为进一步研究B19病毒VP1u在病毒复制周期的作用奠定基础。 相似文献
17.
P Lemarchand M Vaglio J Mau?l M Markert 《The Journal of biological chemistry》1992,267(27):19379-19382
To further understand the mechanisms involved in phagocyte activation in general and in NADPH oxidase activation in particular, a polyclonal antibody was raised in rabbit against a partially purified oxidase preparation. The enzyme was solubilized from zymosan-activated human neutrophils and resting cells and separated by preparative isoelectric focusing electrophoresis. A polyclonal antibody was raised in rabbit against the pI 5.0 fraction, which had the maximum superoxide-producing capacity. Analysis of the polyclonal antibody revealed marked differences between activated and resting neutrophils. The antibody recognized in particular an 8-kDa protein (p8) in resting human neutrophil cytosol and in the membrane of zymosan-activated cells. A polyclonal antibody (anti-p8) was raised against the pure cytosolic p8 protein. This anti-p8 reacted not only with p8, but also with cytosolic proteins of 14 kDa and 6 kDa. N-terminal amino acid sequence analysis of p8 revealed homology with the calcium-binding myeloid related protein (MRP-8). Upon neutrophil activation, translocation of the 8- and 14-kDa proteins to the membrane was observed with stimuli known to depend on extracellular calcium. In calcium-depleted medium, the absence of translocation correlated with a depression of superoxide production, supporting a role for the calcium-binding protein in cellular activation. 相似文献
18.
A temperature-sensitive mutant of 3Y1, 3Y1tsD123, reversibly arrested in G1 phase of cell cycle at the restrictive temperature of 39.8 degrees C, shows a single amino acid exchange in the D123 protein. In this study, we found that the D123 protein level in 3Y1tsD123, which was 1/8 of that in 3Y1 compared at the permissive temperature of 33.9 degrees C, lowered to 1/4 after a shift to the restrictive temperature. During inhibition of protein synthesis with cycloheximide, the D123 protein level in 3Y1tsD123 decreased markedly depending on the incubation temperature, compared with that in 3Y1, indicating that the lowered levels of D123 protein in 3Y1tsD123 are due to its degradation. Unexpectedly, 2 stably temperature-resistant clones were isolated after transfection of SV-3Y1tsD123 (SV40-transformed 3Y1tsD123, which shows cell death instead of G1 arrest at the restrictive temperature) with the cDNA of the mutant-type (3Y1tsD123-derived) D123 protein. The D123 protein in both clones degraded extensively at both temperatures, suggesting that the overexpression of the mutant-type D123 protein exceeds its degradation. Both temperature-resistant clones contained higher levels of D123 protein at the restrictive temperature than did SV-3Y1tsD123 at the permissive temperature. We concluded that the lowered D123 protein level at the restrictive temperature induces the temperature-sensitive characteristics of 3Y1tsD123 and SV-3Y1tsD123. 相似文献
19.
Angelika F. Perzlmaier Frank Richter Wolfgang Seufert 《The Journal of biological chemistry》2013,288(30):21537-21546
The eukaryotic translation initiation factor 2 (eIF2) is central to the onset of protein synthesis and its modulation in response to physiological demands. eIF2, a heterotrimeric G-protein, is activated by guanine nucleotide exchange to deliver the initiator methionyl-tRNA to the ribosome. Here we report that assembly of the eIF2 complex in vivo depends on Cdc123, a cell proliferation protein conserved among eukaryotes. Mutations of CDC123 in budding yeast reduced the association of eIF2 subunits, diminished polysome levels, and increased GCN4 expression indicating that Cdc123 is critical for eIF2 activity. Cdc123 bound the unassembled eIF2γ subunit, but not the eIF2 complex, and the C-terminal domain III region of eIF2γ was both necessary and sufficient for Cdc123 binding. Alterations of the binding site revealed a strict correlation between Cdc123 binding, the biological function of eIF2γ, and its ability to assemble with eIF2α and eIF2β. Interestingly, high levels of Cdc123 neutralized the assembly defect and restored the biological function of an eIF2γ mutant. Moreover, the combined overexpression of eIF2 subunits rescued an otherwise inviable cdc123 deletion mutant. Thus, Cdc123 is a specific eIF2 assembly factor indispensable for the onset of protein synthesis. Human Cdc123 is encoded by a disease risk locus, and, therefore, eIF2 biogenesis control by Cdc123 may prove relevant for normal cell physiology and human health. This work identifies a novel step in the eukaryotic translation initiation pathway and assigns a biochemical function to a protein that is essential for growth and viability of eukaryotic cells. 相似文献
20.
A membrane glycoprotein, Sec12p, required for protein transport from the endoplasmic reticulum to the Golgi apparatus in yeast 总被引:47,自引:16,他引:31
下载免费PDF全文
![点击此处可从《The Journal of cell biology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
SEC12, a gene that is required for secretory, membrane, and vacuolar proteins to be transported from the endoplasmic reticulum to the Golgi apparatus, has been cloned from a genomic library by complementation of a sec12 ts mutation. Genetic analysis has shown that the cloned gene integrates at the SEC12 locus and that a null mutation at the locus is lethal. The DNA sequence predicts a protein of 471 amino acids containing a hydrophobic stretch of 19 amino acids near the COOH terminus. To characterize the gene product (Sec12p) in detail, a lacZ-SEC12 gene fusion has been constructed and a polyclonal antibody raised against the hybrid protein. The antibody recognizes Sec12p as a approximately 70-kD protein that sediments in a mixed membrane fraction that includes endoplasmic reticulum. Sec12p is not removed from the membrane fraction by treatment at high pH and high salt and is not degraded by exogenous protease unless detergent is present. Glycosylation of Sec12p during biogenesis is indicated by an electrophoretic mobility shift of the protein that is influenced by tunicamycin and by imposition of an independent secretory pathway block. We suggest that Sec12p is an integral membrane glycoprotein with a prominent domain that faces the cytoplasm where it functions to promote protein transport to the Golgi apparatus. In the process of transport, Sec12p itself may migrate to the Golgi apparatus and function in subsequent transport events. 相似文献