Extracting sequence motifs and the phylogenetic features of SNARE-dependent membrane traffic

The SNARE proteins are required for membrane fusion during intracellular vesicular transport and for its specificity. Only the unique combination of SNARE proteins (cognates) can be bound and can lead to membrane fusion, although the characteristics of the possible specificity of the binding combinations encoded in the SNARE sequences have not yet been determined. We discovered by whole genome sequence analysis that sequence motifs (conserved sequences) in the SNARE motif domains for each protein group correspond to localization sites or transport pathways. We claim that these motifs reflect the specificity of the binding combinations of SNARE motif domains. Using these motifs, we could classify SNARE proteins from 48 organisms into their localization sites or transport pathways. The classification result shows that more than 10 SNARE subgroups are kingdom specific and that the SNARE paralogs involved in the plasma membrane-related transport pathways have developed greater variations in higher animals and higher plants than those involved in the endoplasmic reticulum-related transport pathways throughout eukaryotic evolution.     

Endosome-to-Golgi transport pathways in physiological processes

The trans-Golgi network (TGN) is a major traffic hub of the cell, as it regulates membrane transport in the secretory pathway as well as receiving protein cargo by retrograde transport from endocytic compartments. Retrograde transport between endosomes and the TGN is essential for the recycling of membrane proteins which regulate a range of cellular and development functions. In addition, retrograde transport pathways are exploited by many bacterial toxins to mediate cytotoxicity and by some viral proteins to promote pathogenicity. Recent advances using a range of molecular cell biological strategies have identified multiple retrograde transport pathways each regulated by a distinct set of molecular machinery. Here we review recent advances in this field and highlight the importance of these transport pathways in a range of physiological processes.     

Protein transport in Archaea: Sec and twin arginine translocation pathways

The transport of proteins into and across hydrophobic membranes is an essential cellular process. The majority of proteins that are translocated in an unfolded conformation traverse the membrane by way of the universally conserved Sec pathway, whereas the twin arginine translocation pathway is responsible for the transport of folded proteins across the membrane. Structural, biochemical and genetic analyses of these processes in Archaea have revealed unique archaeal features, and have also provided a better understanding of these pathways in organisms of all domains. Further study of these pathways in Archaea might elucidate fundamental principles involved in each type of transport and could help determine their relative costs and benefits as well as evolutionary adaptations in protein secretion strategies.     

Protein Traffic in Gram-negative bacteria - how exported and secreted proteins find their way

Gram-negative bacteria assemble many proteins into the inner and outer membranes and export a large number of proteins to the periplasm or to the extracellular medium. During the billions of years bacteria have been around, they have evolved a number of different pathways with sophisticated machines to accurately and efficiently move proteins from one location to another. In this review, we first introduce specific proteins that are representative substrates of the protein transport pathways and describe their function. Then, their specific routes from synthesis to their destinations are described mentioning the signal peptide that may initiate their export and discuss what is known about the folding state of the substrates during transport. The membrane translocation device involved, the energy source required for transport, and whether a chaperone is needed will be discussed.     

Biological Significance of the Importin‐β Family‐Dependent Nucleocytoplasmic Transport Pathways

Importin‐β family proteins (Imp‐βs) are nucleocytoplasmic transport receptors (NTRs) that import and export proteins and RNAs through the nuclear pores. The family consists of 14–20 members depending on the biological species, and each member transports a specific group of cargoes. Thus, the Imp‐βs mediate multiple, parallel transport pathways that can be regulated separately. In fact, the spatiotemporally differential expressions and the functional regulations of Imp‐βs have been reported. Additionally, the biological significance of each pathway has been characterized by linking the function of a member of Imp‐βs to a cellular consequence. Connecting these concepts, the regulation of the transport pathways conceivably induces alterations in the cellular physiological states. However, few studies have linked the regulation of an importin‐β family NTR to an induced cellular response and the corresponding cargoes, despite the significance of this linkage in comprehending the biological relevance of the transport pathways. This review of recent reports on the regulation and biological functions of the Imp‐βs highlights the significance of the transport pathways in physiological contexts and points out the possibility that the identification of yet unknown specific cargoes will reinforce the importance of transport regulation.      

Intracellular sorting and transport of proteins

The secretory and endocytic pathways of eukaryotic organelles consist of multiple compartments, each with a unique set of proteins and lipids. Specific transport mechanisms are required to direct molecules to defined locations and to ensure that the identity, and hence function, of individual compartments are maintained. The localisation of proteins to specific membranes is complex and involves multiple interactions. The recent dramatic advances in understanding the molecular mechanisms of membrane transport has been due to the application of a multi-disciplinary approach, integrating membrane biology, genetics, imaging, protein and lipid biochemistry and structural biology. The aim of this review is to summarise the general principles of protein sorting in the secretory and endocytic pathways and to highlight the dynamic nature of these processes. The molecular mechanisms involved in this transport along the secretory and endocytic pathways are discussed along with the signals responsible for targeting proteins to different intracellular locations.     

Rab proteins: The key regulators of intracellular vesicle transport

Vesicular/membrane trafficking essentially regulates the compartmentalization and abundance of proteins within the cells and contributes in many signalling pathways. This membrane transport in eukaryotic cells is a complex process regulated by a large and diverse array of proteins. A large group of monomeric small GTPases; the Rabs are essential components of this membrane trafficking route. Most of the Rabs are ubiquitously expressed proteins and have been implicated in vesicle formation, vesicle motility/delivery along cytoskeleton elements and docking/fusion at target membranes through the recruitment of effectors. Functional impairments of Rabs affecting transport pathways manifest different diseases. Rab functions are accompanied by cyclical activation and inactivation of GTP-bound and GDP-bound forms between the cytosol and membranes which is regulated by upstream regulators. Rab proteins are characterized by their distinct sub-cellular localization and regulate a wide variety of endocytic, transcytic and exocytic transport pathways. Mutations of Rabs affect cell growth, motility and other biological processes.     

Models of the Transmembrane Domains of the Yeast Mitochondrial CitrateTransport Protein

We have used cysteine scanning, hydropathy analysis and molecular modeling to construct four possible models of the transmembrane helical domains of the yeast mitochondrial citrate transport protein. Models 1 and 2 invoke the formation of a translocation pathway by the six membrane-spanning alpha-helical domains that comprise each citrate transport protein monomer. Thus the homodimeric CTP (the functional form of the CTP) would contain two separate translocation pathways. Models 3 and 4 explore a novel way in which dimerization might take place, in which transmembrane domain 3 would form part of the dimer interface. This would lead to the formation of two seven-helix translocation pathways within the transporter dimer. Importantly, these studies have led to the construction of the first detailed structural models for any of the mitochondrial anion transport proteins, a family of proteins which is essential to cellular bioenergetics. Furthermore, these models suggest numerous experiments which can be carried out to further elucidate the structure of the translocation pathway through the membrane.     

Energy use by biological protein transport pathways

The targeting of proteins into and across biological membranes to their correct cellular locations is mediated by a variety of transport pathways. These systems must couple the thermodynamically unfavorable processes of substrate translocation and integration with the expenditure of metabolic energy, using the free energy of ATP and GTP hydrolysis and/or a transmembrane protonmotive force. Several recent advances in our knowledge of the structure and function of these transport systems have provided insights into the mechanisms of energy transduction, force generation and energy use by different protein transport pathways.     

Recognition and delivery of effector proteins into eukaryotic cells by bacterial secretion systems

The direct transport of virulence proteins from bacterium to host has emerged as a common strategy employed by Gram-negative pathogens to establish infections. Specialized secretion systems function to facilitate this process. The delivery of 'effector' proteins by these secretion systems is currently confined to two functionally similar but mechanistically distinct pathways, termed type III and type IV secretion. The type III secretion pathway is ancestrally related to the multiprotein complexes that assemble flagella, whereas the type IV mechanism probably emerged from the protein complexes that support conjugal transfer of DNA. Although both pathways serve to transport proteins from the bacterium to host, the recognition of the effector protein substrates and the secretion information contained in these proteins appear highly distinct. Here, we review the mechanisms involved in the selection of substrates by each of these transport systems and secretion signal information required for substrate transport.     

Metabolic engineering of plants: the role of membrane transport.

As plant cells are highly compartmentalized, the entrance and exit points of metabolic pathways frequently involve membrane passages of solutes. Transport proteins are often located in strategic positions to control whole pathways and have to be considered in the development of metabolic engineering strategies. Here, we discuss examples of pathways (in carbohydrate metabolism, amino acid and secondary compound synthesis, and mineral metabolism) in which membrane transport steps are considered to exert major control and in which transport proteins have been employed to manipulate metabolic fluxes.     

Apical membrane proteins are transported in distinct vesicular carriers

The function of polarized epithelial cells and neurons is achieved through intracellular sorting mechanisms that recognize classes of proteins in the trans-Golgi network (TGN) and deliver them into separate vesicles for transport to the correct surface domain. Some proteins are delivered to the apical membrane after their association with membrane detergent-insoluble glycophosphatidylinositol/cholesterol (DIG) membrane microdomains [1], while some do not associate with DIGs [2-4]. However, it is not clear if this represents transport by two different pathways or if it can be explained by differences in the affinity of individual proteins for DIGs. Here, we investigate the different trafficking mechanisms of two apically sorted proteins, the DIG-associated sucrase-isomaltase (SI) and lactase-phlorizin hydrolase, which uses a DIG-independent pathway [5]. These proteins were tagged with YFP or CFP, and their trafficking in live cells was visualized using confocal laser microscopy. We demonstrate that each protein is localized to distinct subdomains in the same transport vesicle. A striking triangular pattern of concentration of the DIG-associated SI in subvesicular domains was observed. The original vesicles partition into smaller carriers containing either sucrase-isomaltase or lactase-phlorizin hydrolase, but not both, demonstrating for the first time a post-TGN segregation step and transport of apical proteins in different vesicular carriers.     

Motor-cargo interactions: the key to transport specificity

Eukaryotic cells organize their cytoplasm by moving different organelles and macromolecular complexes along microtubules and actin filaments. These movements are powered by numerous motor proteins that must recognize their respective cargoes in order to function. Recently, several proteins that interact with motors have been identified by yeast two-hybrid and biochemical analyses, and their roles in transport are now being elucidated. In several cases, analysis of the binding partners helped to identify new transport pathways, new types of cargo, and transport regulated at the level of motor-cargo binding. We discuss here how different motors of the kinesin, dynein and myosin families recognize their cargo and how motor-cargo interactions are regulated.     

Multiple pathways for sorting mitochondrial precursor proteins

Mitochondria import hundreds of different precursor proteins from the cytosol. More than 50% of mitochondrial proteins do not use the classical import pathway that is guided by amino-terminal presequences, but instead contain different types of internal targeting signals. Recent studies have revealed an unexpected complexity of the mitochondrial protein import machinery and have led to the discovery of new transport pathways. Here, we review the versatility of mitochondrial protein import and its connection to mitochondrial morphology, redox regulation and energetics.     

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.     

Linkers, packages and pathways: new concepts in axonal transport

The molecular mechanisms that generate efficient and directed transport of proteins and organelles in axons remain poorly understood. In the past year, many studies have identified specific transmembrane or scaffold proteins that might link motor proteins to their cargoes. These studies have also identified previously unsuspected pathways and raised the intriguing possibility that pre-packaged groups of functionally related proteins are transported together in the axon. Evidence suggests that fast molecular motor proteins have a role in slow axonal transport, and the axonal transport machinery has been implicated in the genesis of neurodegenerative diseases.     

Toc, Tic, Tat et al.: structure and function of protein transport machineries in chloroplasts

The chloroplast is an organelle of prokaryotic origin that is situated in an eukaryotic cellular environment. As a result of this formerly endosymbiotic situation, the chloroplast houses a unique set of protein transport machineries. Among those are evolutionarily young transport pathways which are responsible for the import of the nuclear-encoded proteins into the organelle as well as ancient pathways operating in the 'export' of proteins from the stroma (the former cyanobacterial cytosol) across the thylakoid membrane into the thylakoid lumen. In this review, we have tried to address the main features of these various transport pathways.     

Identification of genes involved in salt adaptation in the archaeon Methanosarcina mazei Gö1 using genome-wide gene expression profiling

Methanosarcina mazei is a nonhalophilic methanogen that can adapt to 800 mM NaCl. Microarray studies have been used to examine the effect of elevated salinities on the regulation of gene expression in M. mazei. Eighty-four genes of different functional categories, such as solute transport and biosynthesis, Na(+) export, stress response, ion, protein and phosphate transport, metabolic enzymes, regulatory proteins, DNA-modification systems, and cell-surface modulators, were found to be stronger expressed at high salinities. Moreover, 10 genes encoding different metabolic functions including potassium uptake and ATP synthesis were reduced in expression under high salt. The overall expression profiles suggest that M. mazei is able to adapt to high salinities by multiple upregulation of many different cellular functions including protective pathways such as solute transport and biosynthesis, import of phosphate, export of Na(+), and upregulation of pathways for modification of DNA and cell surface architecture.     

Dephosphorylation-dependent sorting of SR splicing factors during mRNP maturation

SR proteins are a family of sequence-specific RNA binding proteins originally discovered as essential factors for pre-mRNA splicing and recently implicated in mRNA transport, stability, and translation. Here, we used a genetic complementation system derived from conditional knockout mice to address the function and regulation of SR proteins in vivo. We demonstrate that ASF/SF2 and SC35 are each required for cell viability, but, surprisingly, the effector RS domain of ASF/SF2 is dispensable for cell survival in MEFs. Although shuttling SR proteins have been implicated in mRNA export, prevention of ASF/SF2 from shuttling had little impact on mRNA export. We found that shuttling and nonshuttling SR proteins are segregated in an orderly fashion during mRNP maturation, indicating distinct recycling pathways for different SR proteins. We further showed that this process is regulated by differential dephosphorylation of the RS domain, thus revealing a sorting mechanism for mRNP transition from splicing to export.     

Membrane tethering and fusion in the secretory and endocytic pathways

Studies of intracellular trafficking over the past decade or so have led to striking advances in our understanding of the molecular processes by which transport intermediates dock and fuse. SNARE proteins play a central role, assembling into complexes that bridge membranes and may catalyze membrane fusion directly. In general, different SNARE proteins operate in different intracellular trafficking pathways, so recent reports that SNARE assembly in vitro is promiscuous have come as something of a surprise. We propose a model in which proper SNARE assembly is under kinetic control, orchestrated by members of the Sec1 protein family, small GTP-binding Rab proteins, and a diverse assortment of tethering proteins.