Model for membrane organization and protein sorting in the cyanobacterium Synechocystis sp. PCC 6803 inferred from proteomics and multivariate sequence analyses

Cyanobacteria are unique eubacteria with an organized subcellular compartmentalization of highly differentiated internal thylakoid membranes (TM), in addition to the outer and plasma membranes (PM). This leads to a complicated system for transport and sorting of proteins into the different membranes and compartments. By shotgun and gel-based proteomics of plasma and thylakoid membranes from the cyanobacterium Synechocystis sp. PCC 6803, a large number of membrane proteins were identified. Proteins localized uniquely in each membrane were used as a platform describing a model for cellular membrane organization and protein intermembrane sorting and were analyzed by multivariate sequence analyses to trace potential differences in sequence properties important for insertion and sorting to the correct membrane. Sequence traits in the C-terminal region, but not in the N-terminal nor in any individual transmembrane segments, were discriminatory between the TM and PM classes. The results are consistent with a contact zone between plasma and thylakoid membranes, which may contain short-lived "hemifusion" protein traffic connection assemblies. Insertion of both integral and peripheral membrane proteins is suggested to occur through common translocons in these subdomains, followed by a potential translation arrest and structure-based sorting into the correct membrane compartment.     

Alteration of N-terminal residues of mature human lysozyme affects its secretion in yeast and translocation into canine microsomal vesicles

Signal sequences play a central role in the initial membrane translocation of secretory proteins. Their functions depend on factors such as hydrophobicity and conformation of the signal sequences themselves. However, some characteristics of mature proteins, especially those of the N-terminal region, might also affect the function of the signal sequences. To examine this possibility, several mutants of human lysozyme modified in the N-terminal region of the mature protein were constructed, and their secretion in yeast as well as in vitro translocation into canine pancreatic microsomes were analyzed using an idealized signal sequence L8 (MR(L)8PLAALG). Our results show the following. (1) Change in the charge at the N-terminal residue of the mature protein does not affect secretion drastically. (2) Substitution of a proline residue at the N terminus prevents cleavage of the signal sequence, although translocation itself is not impaired. (3) Excessive positive charges in the N-terminal region delay translocation of the precursor protein across the membrane. (4) Polar and negatively charged residues introduced into the N-terminal region affect the secretion of the mature protein by preventing its correct folding.     

Expert system for predicting protein localization sites in gram-negative bacteria

We have developed an expert system that makes use of various kinds of knowledge organized as "if-then" rules for predicting protein localization sites in Gram-negative bacteria, given the amino acid sequence information alone. We considered four localization sites: the cytoplasm, the inner (cytoplasmic) membrane, the periplasm, and the outer membrane. Most rules were derived from experimental observations. For example, the rule to recognize an inner membrane protein is the presence of either a hydrophobic stretch in the predicted mature protein or an uncleavable N-terminal signal sequence. Lipoproteins are first recognized by a consensus pattern and then assumed present at either the inner or outer membrane. These two possibilities are further discriminated by examining an acidic residue in the mature N-terminal portion. Furthermore, we found an empirical rule that periplasmic and outer membrane proteins were successfully discriminated by their different amino acid composition. Overall, our system could predict 83% of the localization sites of proteins in our database.     

MotX and MotY,specific components of the sodium-driven flagellar motor,colocalize to the outer membrane in Vibrio alginolyticus

Rotation of the sodium-driven polar flagella of Vibrio alginolyticus requires four motor proteins: PomA, PomB, MotX and MotY. MotX and MotY, which are unique components of the sodium-driven motor of Vibrio, have been believed to be localized in the inner (cytoplasmic) membrane via their N-terminal hydrophobic segments. Here we show that MotX and MotY colocalize to the outer membrane. Both proteins, when expressed together, were detected in the outer membrane fraction separated by sucrose density gradient centrifugation. As mature MotX and MotY proteins do not have N-terminal hydrophobic segments, the N-termini of the primary translation products must have signal sequences that are removed upon translocation across the inner membrane. Moreover, MotX and MotY require each other for efficient localization to the outer membrane. Based on these lines of evidence, we propose that MotX and MotY form a complex in the outer membrane. This is the first case that describes motor proteins function in the outer membrane for flagellar rotation.     

Protein quality control in the bacterial periplasm

The proper functioning of extracytoplasmic proteins requires their export to, and productive folding in, the correct cellular compartment. All proteins in Escherichia coli are initially synthesized in the cytoplasm, then follow a pathway that depends upon their ultimate cellular destination. Many proteins destined for the periplasm are synthesized as precursors carrying an N-terminal signal sequence that directs them to the general secretion machinery at the inner membrane. After translocation and signal sequence cleavage, the newly exported mature proteins are folded and assembled in the periplasm. Maintaining quality control over these processes depends on chaperones, folding catalysts, and proteases. This article summarizes the general principles which control protein folding in the bacterial periplasm by focusing on the periplasmic maltose-binding protein.     

Signal peptide amino acid sequences in Escherichia coli contain information related to final protein localization. A multivariate data analysis

With few exceptions, the signal peptides from proteins inserted into, or translocated through, the membranes of gram-negative bacteria or the endoplasmic reticulum of eukaryotes have no sequence homologies. Therefore these signal peptides have not been considered to contain information related to the different final localizations of the proteins. In this study, 43 signal peptide amino acid sequences from proteins with different final localizations in Escherichia coli have been subjected to a multivariate data analysis. Each amino acid residue was characterized by 20 physico-chemical properties, yielding a multivariate property profile for each peptide. The similarities/dissimilarities in the property profiles for the signal peptides from different classes were compared with each other by generating few-dimensional partial least squares (PLS) discriminant plots. With this approach, signal peptides from proteins localized to the periplasmic space (PS), the outer membrane (OM), and the extracellular surroundings (excreted proteins), were separated into distinct groups. Signal peptides from pili proteins were not separated from the OM signal peptides and only partly from the PS signal peptides, but were clearly different from the signal peptides of the excreted proteins. Signal peptides from inner membrane proteins were similar to those of the PS peptides. The size and the hydrophobicity of different peptide segments were responsible for the separation of the signal peptide classes. For example, the hydrophobicity of the N-terminal segment of the signal peptides increased with an increased distance from the cytoplasm of the final localization for the corresponding proteins. Thus, many signal peptides from proteins with different final localizations in E. coli have different discernible physico-chemical profiles.     

NHE6 protein possesses a signal peptide destined for endoplasmic reticulum membrane and localizes in secretory organelles of the cell

The NHE6 protein is a unique Na(+)/H(+) exchanger isoform believed to localize in mitochondria. It possesses a hydrophilic N-terminal portion that is rich in positively charged residues and many hydrophobic segments. In the present study, signal sequences in the NHE6 molecule were examined for organelle localization and membrane topogenesis. When the full-length protein was expressed in COS7 cells, it localized in the endoplasmic reticulum and on the cell surface. Furthermore, the protein was fully N-glycosylated. When green fluorescent protein was fused after the second (H2) or third (H3) hydrophobic segment, the fusion proteins were targeted to the endoplasmic reticulum (ER) membrane. The localization pattern was the same as that of fusion proteins in which green fluorescent protein was fused after H2 of NHE1. In an in vitro system, H1 behaved as a signal peptide that directs the translocation of the following polypeptide chain and is then processed off. The next hydrophobic segment (H2) halted translocation and eventually became a transmembrane segment. The N-terminal hydrophobic segment (H1) of NHE1 also behaved as a signal peptide. Cell fractionation studies using antibodies against the 15 C-terminal residues indicated that NHE6 protein localized in the microsomal membranes of rat liver cells. All of the NHE6 molecules in liver tissue possess an endoglycosidase H-resistant sugar chain. These findings indicate that NHE6 protein is targeted to the ER membrane via the N-terminal signal peptide and is sorted to organelle membranes derived from the ER membrane.     

An inner membrane protein N-terminal signal sequence is able to promote efficient localisation of an outer membrane protein in Escherichia coli

To test the importance of N-terminal pre-sequences in translocation of different classes of membrane proteins, we exchanged the normal signal sequence of an Escherichia coli outer membrane protein, OmpF, for the pre-sequence of the inner membrane protein, DacA. The DacA-OmpF hybrid was efficiently assembled into the outer membrane in a functionally active form. Thus the pre-sequence of DacA, despite its relatively low hydrophobicity compared with that of OmpF, contains all the essential information necessary to initiate the translocation of OmpF to the outer membrane. Since processing of DacA was also shown to be dependent upon SecA we conclude that the initiation of translocation of this inner membrane polypeptide across the envelope occurs by the same mechanism as outer membrane and periplasmic proteins. The N-terminal 11 amino acids of mature OmpF, which in the hybrid are replaced by the N-terminal nine amino acids of DacA, carry no essential assembly signals since the hybrid protein is apparently assembled with equal efficiency to OmpF.     

The retinal rod Na(+)/Ca(2+),K(+) exchanger contains a noncleaved signal sequence required for translocation of the N terminus

The retinal rod Na(+)/Ca(2+),K(+) exchanger (RodX) is a polytopic membrane protein found in photoreceptor outer segments where it is the principal extruder of Ca(2+) ions during light adaptation. We have examined the role of the N-terminal 65 amino acids in targeting, translocation, and integration of the RodX using an in vitro translation/translocation system. cDNAs encoding human RodX and bovine RodX through the first transmembrane domain were correctly targeted and integrated into microsomal membranes; deletion of the N-terminal 65 amino acids (aa) resulted in a translation product that was not targeted or integrated. Deletion of the first 65 aa had no effect on membrane targeting of full-length RodX, but the N-terminal hydrophilic domain no longer translocated. Chimeric constructs encoding the first 65 aa of bovine RodX fused to globin were translocated across microsomal membranes, demonstrating that the sequence could function heterologously. Studies of fresh bovine retinal extracts demonstrated that the first 65 aa are present in the native protein. These data demonstrate that the first 65 aa of RodX constitute an uncleaved signal sequence required for the efficient membrane targeting and proper membrane integration of RodX.     

Topogenesis of membrane proteins at the endoplasmic reticulum

Most eukaryotic membrane proteins are cotranslationally integrated into the endoplasmic reticulum membrane by the Sec61 translocation complex. They are targeted to the translocon by hydrophobic signal sequences, which induce the translocation of either their N- or their C-terminal sequence. Signal sequence orientation is largely determined by charged residues flanking the apolar sequence (the positive-inside rule), folding properties of the N-terminal segment, and the hydrophobicity of the signal. Recent in vivo experiments suggest that N-terminal signals initially insert into the translocon head-on to yield a translocated N-terminus. Driven by a local electrical potential, the signal may invert its orientation and translocate the C-terminal sequence. Increased hydrophobicity slows down inversion by stabilizing the initial bound state. In vitro cross-linking studies indicate that signals rapidly contact lipids upon entering the translocon. Together with the recent crystal structure of the homologous SecYEbeta translocation complex of Methanococcus jannaschii, which did not reveal an obvious hydrophobic binding site for signals within the pore, a model emerges in which the translocon allows the lateral partitioning of hydrophobic segments between the aqueous pore and the lipid membrane. Signals may return into the pore for reorientation until translation is terminated. Subsequent transmembrane segments in multispanning proteins behave similarly and contribute to the overall topology of the protein.     

Membrane insertion of uracil permease, a polytopic yeast plasma membrane protein.

Uracil permease is a multispanning protein of the Saccharomyces cerevisiae plasma membrane which is encoded by the FUR4 gene and produced in limited amounts. It has a long N-terminal hydrophilic segment, which is followed by 10 to 12 putative transmembrane segments, and a hydrophilic C terminus. The protein carries seven potential N-linked glycosylation sites, three of which are in its N-terminal segment. Overexpression of this permease and specific antibodies were used to show that uracil permease undergoes neither N-linked glycosylation nor proteolytic processing. Uracil permease N-terminal segments of increasing lengths were fused to a reporter glycoprotein, acid phosphatase. The in vitro and in vivo fates of the resulting hybrid proteins were analyzed to identify the first signal anchor sequence of the permease and demonstrate the cytosolic orientation of its N-terminal hydrophilic sequence. In vivo insertion of the hybrid protein bearing the first signal anchor sequence of uracil permease into the endoplasmic reticulum membrane was severely blocked in sec61 and sec62 translocation mutants.     

Essential cytoplasmic domains in the Escherichia coli TatC protein.

The twin-arginine translocation (Tat) system mediates the transport of proteins across the bacterial plasma membrane and chloroplast thylakoid membrane. Operating in parallel with Sec-type systems in these membranes, the Tat system is completely different in both structural and mechanistic terms, and is uniquely able to catalyze the translocation of fully folded proteins across coupled membranes. TatC is an essential, multispanning component that has been proposed to form part of the binding site for substrate precursor proteins. In this study we have tested the importance of conserved residues on the periplasmic and cytoplasmic face of the Escherichia coli protein. We find that many of the mutations on the cytoplasmic face have little or no effect. However, substitution at several positions in the extreme N-terminal cytoplasmic region or the predicted first cytoplasmic loop lead to a significant or complete loss of Tat-dependent export. The mutated strains are unable to grow anaerobically on trimethylamine N-oxide minimal media and are unable to export trimethylamine-N-oxide reductase (TorA). The same mutants are completely unable to export a chimeric protein, comprising the TorA signal peptide linked to green fluorescent protein, indicating that translocation is blocked rather than cofactor insertion into the TorA mature protein. The data point to two essential cytoplasmic domains on the TatC protein that are essential for export.     

Novel bacterial surface display systems based on outer membrane anchoring elements from the marine bacterium Vibrio anguillarum

Surface display of heterologous peptides and proteins such as receptors, antigens, and enzymes on live bacterial cells is of considerable value for various biotechnological and industrial applications. In this study, a series of novel cell surface display systems were examined by using Vibrio anguillarum outer membrane protein and outer membrane lipoprotein as anchoring motifs. These display systems consist of (i) the signal sequence and first 11 N-terminal amino acids of V. anguillarum outer membrane lipoprotein Wza, or the signal sequence and first 9 N-terminal amino acids of the mature major Escherichia coli lipoprotein Lpp, and (ii) transmembrane domains of V. anguillarum outer membrane proteins Omporf1, OmpU, or Omp26La. In order to assay the translocation efficiency of constructed display systems in bacteria, green fluorescent protein (GFP) was inserted to the systems and the results of GFP surface localization confirmed that four of the six surface display systems could successfully display GFP on the E. coli surface. For assaying its potential application in live bacteria carrier vaccines, an excellent display system Wza-Omporf1 was fused with the major capsid protein (MCP) of large yellow croaker iridovirus and introduced into attenuated V. anguillarum strain MVAV6203, and subsequent analysis of MCP surface localization proved that the novel display system Wza-Omporf1 could function as a strong tool in V. anguillarum carrier vaccine development.     

Mapping of export signals of Pseudomonas aeruginosa pilin with alkaline phosphatase fusions.

Pili of Pseudomonas aeruginosa are assembled from monomers of the structural subunit, pilin, after secretion of this protein across the bacterial membrane. These subunits are initally synthesized as precursors (prepilin) with a six-amino-acid leader peptide that is cleaved off during or after membrane traversal, followed by methylation of the amino-terminal phenylalanine residue. This report demonstrates that additional sequences from the N terminus of the mature protein are necessary for membrane translocation. Gene fusions were made between amino-terminal coding sequences of the cloned pilin gene (pilA) and the structural gene for Escherichia coli alkaline phosphatase (phoA) devoid of a signal sequence. Fusions between at least 45 amino acid residues of the mature pilin and alkaline phosphatase resulted in translocation of the fusion proteins across the cytoplasmic membranes of both P. aeruginosa and E. coli strains carrying recombinant plasmids, as measured by alkaline phosphatase activity and Western blotting. Fusion proteins constructed with the first 10 amino acids of prepilin (including the 6-amino-acid leader peptide) were not secreted, although they were detected in the cytoplasm. Therefore, unlike that of the majority of secreted proteins that are synthesized with transient signal sequences, the membrane traversal of pilin across the bacterial membrane requires the transient six-amino-acid leader peptide as well as sequences contained in the N-terminal region of the mature pilin protein.     

Topological characterization of the essential Escherichia coli cell division protein FtsN.

Genetic and biochemical approaches were used to analyze a topological model for FtsN, a 36-kDa protein with a putative transmembrane segment near the N terminus, and to ascertain the requirements of the putative cytoplasmic and membrane-spanning domains for the function of this protein. Analysis of FtsN-PhoA fusions revealed that the putative transmembrane segment of FtsN could act as a translocation signal. Protease accessibility studies of FtsN in spheroblasts and inverted membrane vesicles confirmed that FtsN had a simple bitopic topology with a short cytoplasmic amino terminus, a single membrane-spanning domain, and a large periplasmic carboxy terminus. To ascertain the functional requirements of the N-terminal segments of FtsN, various constructs were made. Deletion of the N-terminal cytoplasmic and membrane-spanning domains led to intracellular localization of the carboxy domain, instability,and loss of function. Replacement of the N-terminal cytoplasmic and membrane-spanning domains with a membrane-spanning domain from MalG restored subcellular localization and function. These N-terminal domains of FtsN could also be replaced by the cleavable MalE signal sequence with restoration of subcellular localization and function. It is concluded that the N-terminal, cytoplasmic, and transmembrane domains of FtsN are not required for function of the carboxy domain other than to transport it to the periplasm. FtsQ and FtsI were also analyzed.     

Novel mechanism of outer membrane targeting of proteins in Gram-negative bacteria

In Gram-negative bacteria, all the proteins destined for the outer membrane are synthesized with a signal sequence that is cleaved, either by the signal peptidase LepB for integral outer membrane proteins or by LspA for lipoproteins, when they cross the cytoplasmic membrane. The Dickeya dadantii protein PnlH does not possess a cleavable signal sequence but is anchored in the outer membrane by an N-terminal targeting signal. Addition of the 41 N-terminal amino acids of PnlH is sufficient for anchoring various hybrid proteins in the outer membrane. This targeting signal presents some of the characteristics of a Tat (twin arginine translocation) signal sequence but without an obvious cleavage site. We found that the Tat translocation pathway is required for the targeting process. This new mechanism of outer membrane protein targeting is probably widespread as PnlH was also addressed to the outer membrane when expressed in Escherichia coli . As PnlH was not detected as a substrate by Tat signal sequence prediction programmes, this would suggest that there may be many other unknown Tat-dependent outer membrane proteins.     

A conserved motif controls nuclear localization of Drosophila Muscleblind

Human Muscleblind-like proteins are alternative splicing regulators that are functionally altered in the RNA-mediated disease myotonic dystrophy. There are different Muscleblind protein isoforms in Drosophila and we previously determined that these have different subcellular localizations in the COS-M6 cell line. Here, we describe the conservation of the sequence motif KRAEK in isoforms C and E and propose a specific function for this motif. Different Muscleblind isoforms localize to the peri-plasma membrane (MblA), cytoplasm (MblB), or show no preference for the nuclear or cytoplasmic compartment (MblC and MblD) in Drosophila S2 cells transiently transfected with Musclebind expression plasmids. Mutation of the KRAEK motif reduces MblC nuclear localization, whereas fusion of a single KRAEK motif to the heterologous protein β-galactosidase is sufficient to target the reporter protein to the nucleus of S2 cells. This motif is not exclusive to Muscleblind proteins and is detected in several other protein types. Taken together, these results suggest that the KRAEK motif regulates nuclear translocation of Muscleblind and may constitute a new class of nuclear localization signal.     

Interactions between Sec complex and prepro-alpha-factor during posttranslational protein transport into the endoplasmic reticulum

Posttranslational translocation of prepro-alpha-factor (ppalphaF) across the yeast endoplasmic reticulum membrane begins with the binding of the signal sequence to the Sec complex, a membrane component consisting of the trimeric Sec61p complex and the tetrameric Sec62p/63p complex. We show by photo-cross-linking that the signal sequence is bound directly to a site where it contacts simultaneously Sec61p and Sec62p, suggesting that there is a single signal sequence recognition step. We found no evidence for the simultaneous contact of the signal sequence with two Sec61p molecules. To identify transmembrane segments of Sec61p that line the actual translocation pore, a late translocation intermediate of ppalphaF was generated with photoreactive probes incorporated into the mature portion of the polypeptide. Cross-linking to multiple regions of Sec61p was observed. In contrast to the signal sequence, neighboring positions of the mature portion of ppalphaF had similar interactions with Sec61p. These data suggest that the channel pore is lined by several transmembrane segments, which have no significant affinity for the translocating polypeptide chain.     

Protein Transport in Plant Cells

The subcellular localization and secretion of proteins synthesized in the cytosol are determined by short amino acid sequences in their molecules. N-terminal transit peptides provide for protein translocation across the membranes of the ER, mitochondria, plastids, and microbodies. Later, these peptides are cleaved off by processing peptidases. C-terminal peptides direct some proteins into microbodies and vacuoles. Transport into the nucleus and insertion in the membranes are determined by the specific sequences that reside in the molecule of the mature protein. Specific receptors associated with the protein-translocating channel recognize transit peptides. Protein unfolding is required for successful protein transport through these channels. Chaperones maintain proteins in such a state. Folded proteins cross the nuclear pore complex and the membrane of microbodies. Protein transport is tightly associated with their processing. During the vesicular protein transport within the endomembrane system (ER, Golgi apparatus, plasma membrane, and vacuoles), correct protein targeting is ensured by protein sorting during vesicle loading, the assembly of corresponding protein coats, vesicle transport to the acceptor membrane, and specific membrane fusion.