Retrograde trafficking and plasma membrane recycling pathways of the budding yeast Saccharomyces cerevisiae

The endosomal system functions as a network of protein and lipid sorting stations that receives molecules from endocytic and secretory pathways and directs them to the lysosome for degradation, or exports them from the endosome via retrograde trafficking or plasma membrane recycling pathways. Retrograde trafficking pathways describe endosome‐to‐Golgi transport while plasma membrane recycling pathways describe trafficking routes that return endocytosed molecules to the plasma membrane. These pathways are crucial for lysosome biogenesis, nutrient acquisition and homeostasis and for the physiological functions of many types of specialized cells. Retrograde and recycling sorting machineries of eukaryotic cells were identified chiefly through genetic screens using the budding yeast Saccharomyces cerevisiae system and discovered to be highly conserved in structures and functions. In this review, we discuss advances regarding retrograde trafficking and recycling pathways, including new discoveries that challenge existing ideas about the organization of the endosomal system, as well as how these pathways intersect with cellular homeostasis pathways.     

Analysis of plant root electropotentials

A model is proposed for the analysis of steady-state electropotentials in plant roots. The radial element of this model is discussed in terms of two parallel pathways, i.e. the symplasm and the extracellular space. The symplasm pathway is treated as a system of three compartments in series. The electrical potential difference across this pathway and its resistance are computed as a function of ion concentrations in the different compartments and the membrane properties. Similarly the extracellular pathway is analyzed as an ion-exchange membrane. Integration of both pathways into a parallel array analog and comparison with experimental data show that the widely used treatment of the root system as a single passive membrane is inadequate. It is also concluded that the occurrence of electrogenic pumps cannot be ascertained by straightforward inhibitor studies. However, the model proved to be insensitive to relatively wide variations in some of the parameters involved in the determination of its behavior.     

Membrane traffic in skeletal muscle

Skeletal muscle tissue is made up of highly organized multinuclear cells. The internal organization of the muscle cell is dictated by the necessary regular arrangement of repeated units within the protein myofibrils that mediate muscle contraction. Skeletal muscle cells have the usual membrane traffic pathways for partitioning newly synthesized proteins, internalizing cell surface receptors for hormones and nutrients, and mediating membrane repair. However, in muscle, these pathways must be further specialized to deal with targeting to and organizing muscle-specific membrane structures, satisfying the unique metabolic requirements of muscle and meeting the high demand for membrane repair in a tissue that is constantly under mechanical stress. Specialized membrane traffic pathways in muscle also play a role in the formation of muscle through fusion of myoblast membranes and the development of internal muscle-specific membrane structures during myogenesis and regeneration. It has recently become apparent that muscle-specific isoforms of proteins that are known to mediate ubiquitous membrane traffic pathways, as well as novel muscle-specific proteins, are involved in tissue-specific aspects of muscle membrane traffic. Here we describe the specialized membrane structures of skeletal muscle, how these are developed, maintained and repaired by specialized and generic membrane traffic pathways, and how defects in these pathways result in muscle disease.     

Pore-forming toxins induce multiple cellular responses promoting survival

Pore-forming toxins (PFTs) are secreted proteins that contribute to the virulence of a great variety of bacterial pathogens. They inflict one of the more disastrous damages a target cell can be exposed to: disruption of plasma membrane integrity. Since this is an ancient form of attack, which bears similarities to mechanical membrane damage, cells have evolved response pathways to these perturbations. Here, it is reported that PFTs trigger very diverse yet specific response pathways. Many are triggered by the decrease in cytoplasmic potassium, which thus emerges as a central regulator. Upon plasma membrane damage, cells activate signalling pathways aimed at restoring plasma membrane integrity and ion homeostasis. Interestingly these pathways do not require protein synthesis. Cells also trigger signalling cascades that allow them to enter a quiescent-like state, where minimal energy is consumed while waiting for plasma membrane damage to be repaired. More specifically, protein synthesis is arrested, cytosolic constituents are recycled by autophagy and energy is stored in lipid droplets.     

Multiple pathways used for the targeting of thylakoid proteins in chloroplasts

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.     

Protein secretion in the absence of ATP: the autotransporter, two-partner secretion and chaperone/usher pathways of gram-negative bacteria (review)

Bacteria secrete a wide variety of proteins, many of which play important roles in virulence. In gram-negative bacteria, these proteins must cross the cytoplasmic or inner membrane, periplasm, and outer membrane to reach the cell surface. Gram-negative bacteria have evolved multiple pathways to allow protein secretion across their complex envelope. ATP is not available in the periplasm and many of these secretion pathways encode components that harness energy available at the inner membrane to drive secretion across the outer membrane. In contrast, the autotransporter, two-partner secretion and chaperone/usher pathways are comparatively simple systems that allow secretion across the outer membrane without the need for input of energy from the inner membrane. This review will present overviews of these 'self-sufficient' pathways, focusing on recent advances and secretion mechanisms. Similarities among the pathways and with other protein translocation mechanisms will be highlighted.     

Protein secretion in the absence of ATP: the autotransporter,two-partner secretion and chaperone/usher pathways of Gram-negative bacteria (Review)

Bacteria secrete a wide variety of proteins, many of which play important roles in virulence. In Gram-negative bacteria, these proteins must cross the cytoplasmic or inner membrane, periplasm, and outer membrane to reach the cell surface. Gram-negative bacteria have evolved multiple pathways to allow protein secretion across their complex envelope. ATP is not available in the periplasm and many of these secretion pathways encode components that harness energy available at the inner membrane to drive secretion across the outer membrane. In contrast, the autotransporter, two-partner secretion and chaperone/usher pathways are comparatively simple systems that allow secretion across the outer membrane without the need for input of energy from the inner membrane. This review will present overviews of these ‘self-sufficient’ pathways, focusing on recent advances and secretion mechanisms. Similarities among the pathways and with other protein translocation mechanisms will be highlighted.     

细胞内吞的研究进展

细胞外基质的各种分子经细胞膜进入真核细胞是一个复杂的过程。细胞内吞是通过细胞质膜的变形运动将细胞外物质转运入细胞内的过程。不同的细胞内吞途径需要不同的蛋白质分子参与,引起不同的信号转导通路。目前认为细胞内吞和膜转运是细胞对其信号转导过程的一种精密的组织安排,细胞内吞在细胞信号转导,维持机体动态平衡方面起着重要作用。细胞内吞途径通常可以分为网格蛋白依赖的内吞和非网格蛋白依赖的内吞,其中后者包括陷窝蛋白依赖和非陷窝蛋白依赖的内吞,以及巨胞饮介导的内吞。本文将就这几种主要细胞内吞途径及与细胞信号转导通路关系的研究进展予以介绍。     

Different activation mechanisms of cystic fibrosis transmembrane conductance regulator expressed in Xenopus laevis oocytes

The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP sensitive Cl- channel that is defective in cystic fibrosis (CF). The most frequent mutation, namely deltaF508-CFTR, accounts for 66% of CF. Here we show that cAMP-activation of CFTR occurs via at least two distinct pathways: activation of CFTR molecules already present in the plasma membrane and protein kinase A (PKA)-mediated vesicular transport of new CFTR molecules to the plasma membrane and functional insertion into the membrane. We investigated the mechanisms that are responsible for these activation pathways using the Xenopus laevis oocytes expression system. We expressed CFTR and recorded continuously membrane current (Im), conductance (Gm) and capacitance (Cm), which is a direct measure of membrane surface area. Expression of CFTR alone did not change the plasma membrane surface area. However, activation of CFTR with cAMP increased Im, Gm and Cm while deltaF508-CFTR-expressing oocytes showed no response on cAMP. Inhibition of protein kinase A or buffering intracellular Ca2+ abolished the cAMP-induced increase in Cm while increases of Im and Gm were still present. ATP or the xanthine derivative 8-cyclopentyl-1,3-dipropylxanthine (CPX) did not further activate CFTR. Insertion of pre-formed CFTR into the plasma membrane could be prevented by compounds that interfere with intracellular transport mechanisms such as primaquine, brefeldin A, nocodazole. From these data we conclude that cAMP activates CFTR by at least two distinct pathways: activation of CFTR already present in the plasma membrane and exocytotic delivery of new CFTR molecules to the oocyte membrane and functional insertion into it.     

Continuity of the Chloroplast Membrane Systems in Zea mays L

Ultrastructural studies of the chloroplasts of the normal, yellow-green, and pale green phenotypes of Zea mays L. indicate that the internal membrane system is continuous with the plastid envelop. The intramembraneous spaces, loculi, and fret channels are also continuous with inner component of the plastid envelop. High energy compounds or other photosynthates, formed in the grana or frets are thus separated from both stroma and cytoplasm by a single membrane, either the fret membrane or the outer component of the plastid envelop. Since this type of plastid ultrastructure is apparently found only in plants exhibiting the Hatch and Slack pathways of photosynthesis there may be a relation between plastid ultrastructure and the pathways of photosynthetic carbon fixation.     

Lipid raft-independent B cell receptor-mediated antigen internalization and intracellular trafficking

The Ag-specific B cell receptor (BCR) expressed by B lymphocytes has two distinct functions upon interaction with cognate Ag: signal transduction (generation of intracellular second messenger molecules) and Ag internalization for subsequent processing and presentation. While it is known that plasma membrane domains, termed lipid rafts, are involved in BCR-mediated signal transduction, the precise role of plasma membrane lipid rafts in BCR-mediated Ag internalization and intracellular trafficking is presently unclear. Using a highly characterized model system, it was determined that while plasma membrane lipid rafts can be internalized by B lymphocytes, lipid rafts do not represent a major pathway for the rapid and efficient internalization of cell surface Ag-BCR complexes. Moreover, internalized plasma membrane lipid rafts are delivered to intracellular compartments distinct from those to which the bulk of internalized Ag-BCR complexes are delivered. These results demonstrate that B lymphocytes, like other cell types, possess at least two distinct endocytic pathways (i.e., clathrin-coated pits and plasma membrane lipid rafts) that deliver internalized ligands to distinct intracellular compartments. Furthermore, Ag-BCR complexes differentially access these two distinct internalization pathways.     

On the biogenesis of myelin membranes: Sorting, trafficking and cell polarity

In the central nervous system, a multilayered membrane layer known as the myelin sheath enwraps axons, and is required for optimal saltatory signal conductance. The sheath develops from membrane processes that extend from the plasma membrane of oligodendrocytes and displays a unique lipid and protein composition. Myelin biogenesis is carefully regulated, and multiple transport pathways involving a variety of endosomal compartments are involved. Here we briefly summarize how the major myelin proteins proteolipid protein and myelin basic protein reach the sheath, and highlight potential mechanisms involved, including the role of myelin specific lipids and cell polarity related transport pathways.     

Metabotropic glutamate receptors promote neuronal and vascular plasticity through novel intracellular pathways

During the initial development and maturation of an individual, the metabotropic glutamate receptor (mGluR) system becomes a necessary component for the critical integration of cellular function and plasticity. In addition to the maintenance of cellular physiology, the mGluR system plays a critical role during acute and chronic degenerative disorders of the central nervous system. By coupling to guanosine-nucleotide-binding proteins (G-proteins), the mGluR system employs a broad range of signal transduction systems to regulate cell survival and injury. More commonly, it is the activation of specific mGluR subtypes that can prevent programmed cell death (PCD) consisting of two distinct pathways of genomic DNA degradation and membrane phosphatidylserine (PS) residue exposure. To offer this cellular protection, mGluRs modulate a series of down-stream cellular pathways that include protein kinases, mitochondrial membrane potential, cysteine proteases, intracellular pH, endonucleases, and mitogen activated protein kinases. Prevention of cellular injury by the mGluR system is directly applicable to clinical disability, since immediate and delayed injury paradigms demonstrate the ability of this system to reverse PCD in both neuronal and vascular cell populations. Further understanding of the intricate pathways that determine the protective nature of the mGluR system will provide new therapeutic avenues for the treatment of neurodegenerative disorders.     

Behaviour of topological marker proteins targeted to the Tat protein transport pathway

The Escherichia coli Tat system mediates Sec-independent export of protein precursors bearing twin arginine signal peptides. Formate dehydrogenase-N is a three-subunit membrane-bound enzyme, in which localization of the FdnG subunit to the membrane is Tat dependent. FdnG was found in the periplasmic fraction of a mutant lacking the membrane anchor subunit FdnI, confirming that FdnG is located at the periplasmic face of the cytoplasmic membrane. However, the phenotypes of gene fusions between fdnG and the subcellular reporter genes phoA (encoding alkaline phosphatase) or lacZ (encoding beta-galactosidase) were the opposite of those expected for analogous fusions targeted to the Sec translocase. PhoA fusion experiments have previously been used to argue that the peripheral membrane DmsAB subunits of the Tat-dependent enzyme dimethyl sulphoxide reductase are located at the cytoplasmic face of the inner membrane. Biochemical data are presented that instead show DmsAB to be at the periplasmic side of the membrane. The behaviour of reporter proteins targeted to the Tat system was analysed in more detail. These data suggest that the Tat and Sec pathways differ in their ability to transport heterologous passenger proteins. They also suggest that caution should be observed when using subcellular reporter fusions to determine the topological organization of Tat-dependent membrane protein complexes.     

Targeting of lumenal proteins across the thylakoid membrane

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.     

Osmomechanical regulation of membrane trafficking in polarized cells

The regulation of membrane trafficking is thought to be predominantly under the control of agonist-receptor transduction pathways. In the present study, osmomechanical stress due to swelling, a condition often accompanying cell activation, was shown to induce multiple membrane trafficking pathways in polarized absorptive epithelial cells in the absence of agonists. Osmomechanical stress activated rapidly (seconds) pathways of calcium-dependent membrane insertion into the basolateral domain, pathways of calcium-independent membrane retrieval from the basolateral domain, and a novel pathway of transcytosis (transcellular) between basolateral and apical cell domains. These pathways appear to underlie the transfer and regulation of transport proteins amongst cell compartments. This broad affect of osmomechanical stress on trafficking pathways may reflect a global mechanism for redistribution of transport proteins and other membrane components amongst cell compartments during states of mechanical stress.     

Membrane-specific targeting of green fluorescent protein by the Tat pathway in the cyanobacterium Synechocystis PCC6803

The transport and sorting of extracytoplasmic proteins in cyanobacteria is made complex by the presence of a highly differentiated membrane system. Proteins destined for the periplasm and thylakoid lumen are initially transported by Sec- and Tat-type pathways but little is known of the mechanisms that ultimately direct them to the correct destinations. We have generated a Synechocystis PCC6803 transformant that expresses a fusion protein comprising the Tat-specific targeting signal of Escherichia coli TorA linked to green fluorescent protein (GFP). Immunoblotting indicates the presence of mature-size GFP but no precursor form, demonstrating that efficient translocation has taken place. Confocal microscopy and immunogold electron microscopy reveal GFP to be almost exclusively located in the periplasm, with almost no protein evident in the thylakoid network. These data point to the operation of highly effective sorting pathways for soluble proteins in this cyanobacterium. The observed sorting of the GFP suggests that either (a) the Tat apparatus is located only in the plasma membrane or (b) the TorA-GFP is targeted across either membrane but the GFP is subsequently directed to the periplasm, perhaps by a default sorting pathway to this compartment.     

NADH-dependent aryl hydrocarbon hydroxylase in rat liver mitochondrial outer membrane.

NADH-dependent 3,4-benzpyrene hydroxylase activity was detected in the purified mitochondrial outer membrane fraction from the livers of rats treated with 3-methylcholanthrene. The specific activity in the outer membrane fraction is nearly equal to that of microsomes, a level too high to be accounted for only by the microsomal contamination. On the other hand, the NADPH-dependent 3,4-benzpyrene hydroxylase activity in the outer membrane fraction is about 50% of that of microsomes. The ratio of the specific activity of NADPH- to NADH-dependent 3,4-benzpyrene hydroxylase in microsomal fraction was about 3.5, while that of the outer membrane fraction was about 1.5. Moreover, it was found that NADH-dependent 3,4-benzpyrene hydroxylase activity in mitochondrial outer membrane from control rat liver was cyanide-insensitive, while that in microsomes was cyanide-sensitive. These results suggest the presence in the mitochondrial outer membrane fraction of aryl hydrocarbon hydroxylase activity which uses as electron donor NADH nearly to the same extent as NADPH. The hydroxylase system is composed of cyanide-insensitive cytochrome P-450 and is inducible markedly by 3-methylcholanthrene treatment. The probable electron transfer pathways in the mitochondrial outer membrane cytochrome P-450 oxidase system are discussed.     

Signal transduction by the polymeric immunoglobulin receptor suggests a role in regulation of receptor transcytosis

Many membrane traffic events that were previously thought to be constitutive recently have been found to be regulated by a variety of intracellular signaling pathways. The polymeric immunoglobulin receptor (pIgR) transcytoses dimeric IgA (dIgA) from the basolateral to the apical surface of polarized epithelial cells. Transcytosis is stimulated by binding of dIgA to the pIgR, indicating that the pIgR can transduce a signal to the cytoplasmic machinery responsible for membrane traffic. We report that dIgA binding to the pIgR causes activation of protein kinase C (PKC) and release of inositol 1,4,5- trisphosphate (IP3). The IP3 causes an elevation of intracellular Ca. Artificially activating PKC with phorbol myristate acetate or poisoning the calcium pump with thapsigargin stimulates transcytosis of pIgR, while the intracellular Ca chelator BAPTA-AM inhibits transcytosis. Our data suggest that ligand-induced signaling by the pIgR may regulate membrane traffic via well-known second messenger pathways involving PKC, IP3, and Ca. This may be a model of a general means by which membrane traffic is regulated by receptor-ligand interaction and signaling pathways.