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1.
The biogenesis of the plant thylakoid network is an enormously complex process in terms of protein targeting. The membrane system contains a large number of proteins, some of which are synthesized within the organelle, while many others are imported from the cytosol. Studies in recent years have shown that the targeting of imported proteins into and across the thylakoid membrane is particularly complex, with four different targeting pathways identified to date. Two of these are used to target membrane proteins: a signal recognition particle (SRP)-dependent pathway and a highly unusual pathway that appears to require none of the known targeting apparatus. Two further pathways are used to translocate lumenal proteins across the thylakoid membrane from the stroma and, again, the two pathways differ dramatically from each other. One is a Sec-type pathway, in which ATP hydrolysis by SecA drives the transport of the substrate protein through the membrane in an unfolded conformation. The other is the twin-arginine translocation (Tat) pathway, where substrate proteins are transported in a folded state using a unique mechanism that harnesses the proton motive force across the thylakoid membrane. This article reviews progress in studies on the targeting of lumenal proteins, with reference to the mechanisms involved, their evolution from endosymbiotic progenitors of the chloroplast, and possible elements of regulation.  相似文献   

2.
Several positively charged DNA-binding proteins such as the human immunodeficiency virus Tat protein, the Antennapedia (Antp) homeobox protein, and the herpes simplex virus VP22 protein have been reported to translocate across cell membranes and accumulate in cell nuclei. The import occurs by a poorly understood mechanism that appears to be receptor- and energy-independent. We showed that both VP22 and the positively charged histone H1 adhered to the cell membrane of living cells and were not removed by extensive washing. However, after fixation the proteins relocated to the cell nucleus. The nuclear accumulation of VP22 and histone H1 after fixation shows that positively charged proteins may appear to translocate across the cell membrane because of a fixation artifact. The majority of studies on "membrane permeable" proteins and peptides have been performed using fixation techniques, and our study shows that influx of these proteins may occur during fixation rather than in living cells.  相似文献   

3.
All living organisms, no matter how simple or complex, possess the ability to translocate proteins across biological membranes and into different cellular compartments. Although a range of membrane transport processes exist, the major pathway used to translocate proteins across the bacterial cytoplasmic membrane or the eukaryotic endoplasmic reticulum membrane is conserved and is known as the Sec or Sec61 pathway, respectively. Over the past two decades the Sec and Sec61 pathways have been studied extensively and are well characterised at the genetic and biochemical levels. However, it is only now with the recent structural determination of a number of the key elements of the pathways that the translocation complex is beginning to give up its secrets in exquisite molecular detail. This article will focus on the routes of Sec- and Sec61-dependent membrane targeting and the nature of the translocation channel in bacteria and eukaryotes.  相似文献   

4.
All living organisms, no matter how simple or complex, possess the ability to translocate proteins across biological membranes and into different cellular compartments. Although a range of membrane transport processes exist, the major pathway used to translocate proteins across the bacterial cytoplasmic membrane or the eukaryotic endoplasmic reticulum membrane is conserved and is known as the Sec or Sec61 pathway, respectively. Over the past two decades the Sec and Sec61 pathways have been studied extensively and are well characterised at the genetic and biochemical levels. However, it is only now with the recent structural determination of a number of the key elements of the pathways that the translocation complex is beginning to give up its secrets in exquisite molecular detail. This article will focus on the routes of Sec- and Sec61-dependent membrane targeting and the nature of the translocation channel in bacteria and eukaryotes.  相似文献   

5.
The assembly of the chloroplast thylakoid membrane requires the import of numerous proteins from the cytosol and their targeting into or across the thylakoid membrane. It is now clear that multiple pathways are involved in the thylakoid-targeting stages, depending on the type of protein substrate. Two very different pathways are used by thylakoid lumen proteins; one is the Sec pathway which has been well-characterised in bacteria, and which involves the threading of the substrate through a narrow channel. In contrast, the more recently characterised twin-arginine translocation (Tat) system is able to translocate fully folded proteins across this membrane. Recent advances on bacterial Tat systems shed further light on the structure and function of this system. Membrane proteins, on the other hand, use two further pathways. One is the signal recognition particle-dependent pathway, involving a complex interplay between many different factors, whereas other proteins insert without the assistance of any known apparatus. This article reviews advances in the study of these pathways and considers the rationale behind the surprising complexity.  相似文献   

6.
Proteins that are able to translocate across biological membranes assume a loosely folded structure. In this review it is suggested that the loosely folded structure, referred to here as the 'pre-folded conformation', is a particular structure that interacts favourably with components of the export apparatus. Two soluble factors, SecB and GroEL, have been implicated in maintenance of the pre-folded conformation and have been termed 'molecular chaperones'. Results suggest that SecB may be a chaperone that is specialized for binding to exported protein precursors, while GroEL may be a general folding modulator that binds to many intracellular proteins.  相似文献   

7.
In bacteria, phospholipids are synthesized on the inner leaflet of the cytoplasmic membrane and must translocate to the outer leaflet to propagate a bilayer. Transbilayer movement of phospholipids has been shown to be fast and independent of metabolic energy, and it is predicted to be facilitated by membrane proteins (flippases) since transport across protein-free membranes is negligible. However, it remains unclear as to whether proteins are required at all and, if so, whether specific proteins are needed. To determine whether bacteria contain specific proteins capable of translocating phospholipids across the cytoplasmic membrane, we reconstituted a detergent extract of Bacillus subtilis into proteoliposomes and measured import of a water-soluble phospholipid analog. We found that the proteoliposomes were capable of transporting the analog and that transport was inhibited by protease treatment. Active proteoliposome populations were also able to translocate a long-chain phospholipid, as judged by a phospholipase A(2)-based assay. Protein-free liposomes were inactive. We show that manipulation of the reconstitution mixture by prior chromatographic fractionation of the detergent extract, or by varying the protein/phospholipid ratio, results in populations of vesicles with different specific activities. Glycerol gradient analysis showed that the majority of the transport activity sedimented at approximately 4S, correlating with the presence of specific proteins. Recovery of activity in other gradient fractions was low despite the presence of a complex mixture of proteins. We conclude that bacteria contain specific proteins capable of facilitating transbilayer translocation of phospholipids. The reconstitution methodology that we describe provides the basis for purifying a facilitator of transbilayer phospholipid translocation in bacteria.  相似文献   

8.
9.
Requirements for the membrane insertion of signal-anchor type proteins   总被引:16,自引:5,他引:11       下载免费PDF全文
Proteins which are inserted and anchored in the membrane of the ER by an uncleaved signal-anchor sequence can assume two final orientations. Type I signal-anchor proteins translocate the NH2 terminus across the membrane while type II signal-anchor proteins translocate the COOH terminus. We investigated the requirements for cytosolic protein components and nucleotides for the membrane targeting and insertion of single-spanning type I signal-anchor proteins. Besides the ribosome, signal recognition particle (SRP), GTP, and rough microsomes (RMs) no other components were found to be required. The GTP analogue GMPPNP could substitute for GTP in supporting the membrane insertion of IMC-CAT. By using a photocrosslinking assay we show that for secreted, type I and type II signal-anchor proteins the presence of both GTP and RMs is required for the release of the nascent chain from the 54-kD subunit of SRP. For two of the proteins studied the release of the nascent chain from SRP54 was accompanied by a new interaction with components of the ER. We conclude that the GTP-dependent release of the nascent chain from SRP54 occurs in an identical manner for each of the proteins studied.  相似文献   

10.
赵留群  张大伟 《微生物学报》2022,62(12):4769-4780
依赖信号识别颗粒(signal recognition particle,SRP)的共翻译转运是所有生命体中的一个保守途径,它将新生肽链的翻译与转运耦联在一起。超过30%的新合成的多肽链被SRP转运到正确位置。最近的研究表明,大肠杆菌中SRP抑制子可以规避SRP的需求。当SRP缺失时,翻译控制在介导膜蛋白定位方面起着关键作用。本综述总结了SRP底物如何在存在或缺失SRP的情况下转运到适当的位置以及翻译速率降低如何补偿SRP的缺失。我们还讨论了不同蛋白质对SRP的依赖程度。这一回顾将为进一步研究SRP功能及膜蛋白定位提供新思路。  相似文献   

11.
Several approaches are currently being taken to elucidate the mechanisms and the molecular components responsible for protein targeting to and translocation across the membrane of the endoplasmic reticulum. Two experimental systems dominate the field: a biochemical system derived from mammalian exocrine pancreas, and a combined genetic and biochemical system employing the yeast, Saccharomyces cerevisiae. Results obtained in each of these systems have contributed novel, mostly non-overlapping information. Recently, much effort in the field has been dedicated to identifying membrane proteins that comprise the translocon. Membrane proteins involved in translocation have been identified both in the mammalian system, using a combination of crosslinking and reconstitution approaches, and in S. cerevisiae, by selecting for mutants in the translocation pathway. None of the membrane proteins isolated, however, appears to be homologous between the two experimental systems. In the case of the signal recognition particle, the two systems have converged, which has led to a better understanding of how proteins are targeted to the endoplasmic reticulum membrane.  相似文献   

12.
Mitochondria import the majority of their proteins from the cytosol. At the mitochondrial outer membrane, import is initiated through a series of reactions, which include preprotein recognition, unfolding, insertion and translocation. These processes are facilitated by a multisubunit complex, the TOM complex. Specific roles can now be assigned to several components of this complex. Although the import machinery of the outer membrane can insert and translocate a few proteins on its own, completion of translocation o f most preproteins is dependent upon coupling to both the membrane potential and mt-Hsp70/ATP-driven transport across the inner membrane, mediated by the TIM complex.  相似文献   

13.
The mitochondrial inner membrane of Saccharomyces cerevisiae contains a group of homologous carrier proteins that mediate the exchange of several metabolites. The members of this protein family are synthesized in the cytosol and reach their final topology after translocation across the mitochondrial outer membrane. Using the ADP/ATP carrier (AAC) as a model protein, previous studies have established four distinct steps of the import pathway (stages I-IV). In the absence of the mitochondrial membrane potential (deltapsi), the AAC accumulates at the inner surface of the outer membrane (stage IIIa) and remains bound to the outer membrane import channel. Only in the presence of the membrane potential, can a complex of small Tim proteins mediate transfer of the AAC to the inner membrane. In this study, we characterized the import pathway of the dicarboxylate carrier (DIC). Different from the AAC, the DIC showed complete deltapsi-independent translocation across the outer membrane, release from the import pore, and mainly accumulated in a soluble state in the intermembrane space, thus defining a new translocation intermediate (stage III*). The DIC should be a suitable model protein for the characterization of deltapsi-independent functions of the intermembrane space Tim proteins.  相似文献   

14.
Lipid modification of proteins and their membrane transport   总被引:1,自引:0,他引:1  
An effective method for artificial attachment of lipid anchors to water-soluble proteins has been developed. To this end, a protein molecule is modified in a system of reversed micelles by a water-insoluble reagent, e.g. fatty acid chloride. Fatty acylated proteins acquire an ability to translocate across lipid membranes and penetrate intact cells. This principle of imparting transmembrane properties to water-soluble proteins makes it possible to realize in vivo a direct transport of antibodies across the hemato-encephalic barrier into the brain and to develop a method for virus suppression by fatty acylated anti-viral antibodies capable of penetrating infected cells. The effect of a drastic increase in the biological activity of exogenous protein factors, e.g. Staphylococcus aureus enterotoxin A, as a result of their artificial fatty acylation has been discovered. The above-mentioned phenomena are discussed in relation to the in vivo data, indicating that post-translational modification of proteins by fatty acids and phospholipids is very widespread in nature and evidently plays an important role in protein transport and sorting. In this connection, lipid modification of proteins is regarded as a possible general step of protein transport in vivo.  相似文献   

15.
In contrast to other organisms, gram-negative bacteria have evolved numerous systems for protein export. Eight types are known that mediate export across or insertion into the cytoplasmic membrane, while eight specifically mediate export across or insertion into the outer membrane. Three of the former secretory pathway (SP) systems, type I SP (ISP, ABC), IIISP (Fla/Path) and IVSP (Conj/Vir), can export proteins across both membranes in a single energy-coupled step. A fourth generalized mechanism for exporting proteins across the two-membrane envelope in two distinct steps (which we here refer to as type II secretory pathways [IISP]) utilizes either the general secretory pathway (GSP or Sec) or the twin-arginine targeting translocase for translocation across the inner membrane, and either the main terminal branch or one of several protein-specific export systems for translocation across the outer membrane. We here survey the various well-characterized protein translocation systems found in living organisms and then focus on the systems present in gram-negative bacteria. Comparisons between these systems suggest specific biogenic, mechanistic and evolutionary similarities as well as major differences.  相似文献   

16.
The Tat protein-export system serves to translocate folded proteins, often containing redox cofactors, across the bacterial inner membrane. Substrate proteins are directed to the Tat apparatus by distinctive N-terminal signal peptides containing a consensus SRRxFLK 'twin-arginine' motif. Here we review recent studies of the Tat system with particular emphasis on the assembly of membrane-bound respiratory complexes. We discuss the connection between Tat targeting and topological organisation of the complexes and consider the role of chaperone proteins in cofactor insertion and Tat targeting. The crystal structure of Escherichia coli formate dehydrogenase-N demonstrates that some Tat substrates are integral membrane proteins. Sequence analysis suggests that one-quarter of all traffic on the E. coli Tat pathway is inner-membrane proteins.  相似文献   

17.
The apparatus that permits protein translocation across the internal thylakoid membranes of chloroplasts is completely unknown, even though these membranes have been the subject of extensive biochemical analysis. We have used a genetic approach to characterize the translocation of Chlamydomonas cytochrome f, a chloroplast-encoded protein that spans the thylakoid once. Mutations in the hydrophobic core of the cytochrome f signal sequence inhibit the accumulation of cytochrome f, lead to an accumulation of precursor, and impair the ability of Chlamydomonas cells to grow photosynthetically. One hydrophobic core mutant also reduces the accumulation of other thylakoid membrane proteins, but not those that translocate completely across the membrane. These results suggest that the signal sequence of cytochrome f is required and is involved in one of multiple insertion pathways. Suppressors of two signal peptide mutations describe at least two nuclear genes whose products likely describe the translocation apparatus, and selected second-site chloroplast suppressors further define regions of the cytochrome f signal peptide.  相似文献   

18.
19.
Gangliosides, complex glycosphingolipids containing sialic acids, have been found to reside in glycosphingolipid-enriched microdomains (GEM) at the plasma membrane. They are synthesized in the lumen of the Golgi complex and appear unable to translocate from the lumenal toward the cytosolic surface of Golgi membrane to access the monomeric lipid transport. As a consequence, they can only leave the Golgi complex via the lumenal surface of transport vesicles. In this work we analyzed the exocytic transport of the disialo ganglioside GD3 from trans-Golgi network (TGN) to plasma membrane in CHO-K1 cells by immunodetection of endogenously synthesized GD3. We found that ganglioside GD3, unlike another luminal membrane-bounded lipid (glycosylphosphatidylinositol-anchored protein), did not partition into GEM domains in the Golgi complex and trafficked from TGN to plasma membrane by a brefeldin A-insensitive exocytic pathway. Moreover, a dominant negative form of Rab11, which prevents exit of vesicular stomatitis virus glycoprotein from the Golgi complex, did not influence the capacity of GD3 to reach the cell surface. Our results strongly support the notion that most ganglioside GD3 traffics from the TGN to the plasma membrane by a non-conventional vesicular pathway where lateral membrane segregation of vesicular stomatitis virus glycoprotein (non-GEM resident) and glycosylphosphatidylinositol-anchored proteins (GEM resident) from GD3 is required before exiting TGN.  相似文献   

20.
We have examined the topology of the yeast arginine permease, a plasma-membrane protein with multiple membrane-spanning domains. Using fusions of the permease with the glycosylatable secreted yeast protein, acid phosphatase, we identified membrane-spanning sequences that can translocate adjacent acid phosphatase across the membrane of the endoplasmic reticulum (ER), as measured by in vitro glycosylation. Examination for the presence or absence of glycosylation in a systematic series of such fusions gave an internally consistent model for the lumenal or cytoplasmic disposition of the acid phosphatase reporter, defining the topology of the permease. The phenotypes of a further set of arginine permease gene fusions with portions of the gene for the secreted protein, killer toxin, suggest that the pathways of export of membrane and secreted proteins need not be functionally distinct.  相似文献   

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