Prokaryotic Cell Membranes Research Articles

Crystal structure of a membrane-embedded H+-translocating pyrophosphatase

H(+)-translocating pyrophosphatases (H(+)-PPases) are active proton transporters that establish a proton gradient across the endomembrane by means of pyrophosphate (PP(i)) hydrolysis. H(+)-PPases are found primarily as homodimers in the vacuolar membrane of plants and the plasma membrane of several protozoa and prokaryotes. The three-dimensional structure and detailed mechanisms underlying the enzymatic and proton translocation reactions of H(+)-PPases are unclear. Here we report the crystal structure of a Vigna radiata H(+)-PPase (VrH(+)-PPase) in complex with a non-hydrolysable substrate analogue, imidodiphosphate (IDP), at 2.35 Å resolution. Each VrH(+)-PPase subunit consists of an integral membrane domain formed by 16 transmembrane helices. IDP is bound in the cytosolic region of each subunit and trapped by numerous charged residues and five Mg(2+) ions. A previously undescribed proton translocation pathway is formed by six core transmembrane helices. Proton pumping can be initialized by PP(i) hydrolysis, and H(+) is then transported into the vacuolar lumen through a pathway consisting of Arg 242, Asp 294, Lys 742 and Glu 301. We propose a working model of the mechanism for the coupling between proton pumping and PP(i) hydrolysis by H(+)-PPases.

Free Energy Difference in Indolicidin Attraction to Eukaryotic and Prokaryotic Model Cell Membranes

We analyzed the thermodynamic and structural determinants of indolicidin interactions with eukaryotic and prokaryotic cell membranes using a series of atomistically detailed molecular dynamics simulations. We used quartz-supported bilayers with two different compositions of zwitterionic and anionic phospholipids as model eukaryotic and prokaryotic cell membranes. Indolicidin was preferentially attracted to the model prokaryotic cell membrane in contrast to the weak adsorption on the eukaryotic membrane. The nature of the indolicidin surface adsorption depended on an electrostatic guiding component, an attractive enthalpic component derived from van der Waals interactions, and a balance between entropic factors related to peptide confinement at the interface and counterion release from the bilayer surface. Thus, whereas we attributed the specificity of the indolicidin/membrane interaction to electrostatics, these interactions were not the sole contributors to the free energy of adsorption. Instead, a balance between an attractive van der Waals enthalpic component and a repulsive entropic component determined the overall strength of indolicidin adsorption.

Mechanisms of Sec61/SecY-Mediated Protein Translocation Across Membranes

The Sec61 or SecY channel, a universally conserved protein-conducting channel, translocates proteins across and integrates proteins into the eukaryotic endoplasmic reticulum (ER) membrane and the prokaryotic plasma membrane. Depending on channel-binding partners, polypeptides are moved by different mechanisms. In cotranslational translocation, the ribosome feeds the polypeptide chain directly into the channel. In posttranslational translocation, a ratcheting mechanism is used by the ER-lumenal chaperone BiP in eukaryotes, and a pushing mechanism is utilized by the SecA ATPase in bacteria. In prokaryotes, posttranslational translocation is facilitated through the function of the SecD/F protein. Recent structural and biochemical data show how the channel opens during translocation, translocates soluble proteins, releases hydrophobic segments of membrane proteins into the lipid phase, and maintains the barrier for small molecules.

Specific lipid recognition is a general feature of CD300 and TREM molecules

CD300, triggering receptor expressed on myeloid cells (TREM), and TREM-like (TREML) receptors are important regulators of the mammalian immune response. Homologs of these receptors, which occur in activating and inhibitory transmembrane forms as well as soluble variants, are found throughout the jawed vertebrates. Specific ligands for most members of these receptor families remain elusive. We report here that at least 11 separate receptors from the CD300, TREM, and TREML families engage in robust and specific interactions with major polar lipids found in prokaryotic and eukaryotic cell membranes. Both soluble and membrane-bound receptor forms exhibit lipid interactions in the solid phase as well as in a physiological signaling context. Overlapping but distinctive patterns of receptor specificity suggest that the CD300/TREM system as a whole may discriminate immunological stimuli based on lipid signatures, thereby influencing downstream responses.

Dynamics of SecY Translocons with Translocation-Defective Mutations

The SecY/Sec61 translocon complex, located in the endoplasmic reticulum membrane of eukaryotes (Sec61) or the plasma membrane of prokaryotes (SecY), mediates the transmembrane secretion or insertion of nascent proteins. Mutations that permit the secretion of nascent proteins with defective signal sequences (Prl-phenotype), or interfere with the transmembrane orientation of newly synthesized protein segments, can affect protein topogenesis. The crystallographic structure of SecYEbeta from Methanococcus jannaschii revealed widespread distribution of mutations causing topogenesis defects, but not their molecular mechanisms. Based upon prolonged molecular dynamics simulations of wild-type M. jannaschii SecYEbeta and an extensive sequence-conservation analysis, we show that the closed state of the translocon is stabilized by hydrogen-bonding interactions of numerous highly conserved amino acids. Perturbations induced by mutation at various locations are rapidly relayed to the plug segment that seals the wild-type closed-state translocon, leading to displacement and increased hydration of the plug.

Para‐aminobenzoic acid is a coenzyme Q biosynthetic precursor in Saccharomyces cerevisiae

Respiratory electron transport in the plasma membrane of prokaryotes and in the inner mitochondrial membrane of eukaryotes is supported by the crucial lipid component, ubiquinone (coenzyme Q or Q). Qn is composed of a fully substituted benzoquinone ring and a polyisoprene tail of n units. The best‐characterized ring precursor, 4‐hydroxybenozoate (4HB), is derived from the chorismate biosynthetic pathway. Extensive work has shown that the ring carbons of 4HB are incorporated into Q6 in yeast, under the activity of an enzyme complex found in the yeast mitochondrial matrix. However, the direct precursor compounds and enzymatic steps for the generation of 4HB in yeast are unknown. Our recent findings show that para‐aminobenzoic acid (pABA) is also a precursor for Q biosynthesis. In yeast pABA is synthesized from chorismate in two steps: Abz1 amidates chorismate to form 4‐deoxyaminochorismate, and Abz2 is a lyase that forms pABA. Here we show that the production of a prenylated form of pABA is dependent on the presence of the ABZ1 gene. We also demonstrate by LC‐MSMS analyses that yeast incubated with stable isotope forms of [13C6‐ring]‐pABA or [13C6‐ring]‐4HB generate [13C6]‐coenzyme Q6. These results identify a novel biosynthetic pathway for the production of Q in yeast. This research was supported by grants from the NIH GM 45952, NSF MCB‐0919609, and by a grant S10RR024605 from the National Center for Research Resources.

Protein translocation through the Sec61/SecY channel

Special codes are embedded in the primary sequence of newly synthesized proteins to determine their final destination. Protein translocation across biological membranes requires co-operation between the targeting and translocation machineries. A conserved membrane channel, the Sec61/SecY complex, mediates protein translocation across or integration into the endoplasmic reticulum membrane in eukaryotes and the plasma membrane in prokaryotes. A combination of recent biochemical and structural data provides novel insights into the mechanism of how the channel allows polypeptide movement into the exoplasmic space and the lipid bilayer.

Three-Dimensional Organization of Chromatids by Nuclear Envelope-Associated Structures

In evolution, the nuclear envelope (NE) arose from the prokaryotic plasma membrane. NE-associated structures, such as nuclear pore complexes (NPCs), the nuclear lamina, and nuclear envelope junctions (NEJs), have evolved to organize, among other things, chromatids within the three-dimensional space of the nucleus. NEJs represent patches of distinct integral membrane proteins of the outer and inner NE membranes, which, by interacting through conserved domains in the perinuclear space, closely align the two NE membranes. In a nuts-and-bolts configuration, the NEJs are linked to repetitive heterochromatin segments of chromatids on their nuclear side and to cytoskeletal elements on their cytoplasmic side. Cytoskeleton-generated mechanical forces are thereby effectively buffered to allow movement of nuclei in the viscous cytoplasm without disrupting the NE. Moreover, these same mechanical forces could generate distortions within the nucleus to facilitate chromatid fluctuations required for DNA repair, replication, and transcription. NPCs are the only route for bidirectional macromolecular transport between the cytoplasm and the nucleus. They also interact with euchromatin segments of chromatids. Thus far, crystallographic analyses of some nucleoporin contact sites suggest considerable plasticity. This flexibility has likely coevolved to not only buffer the mechanical forces propagated from the NEJs to the network of the more than 500 nucleoporins that make up a single NPC, but also impart fluctuations to NPC conformations for transporting large cargoes.

The Endomembrane System: A Representation of the Extracellular Medium?

Both prokaryotic and eukaryotic cells share the basic mechanisms of secretory protein synthesis. However, unlike prokaryotes, eukaryotic cells posses a system of compartments, the so-called endomembrane system, which are involved in the synthesis process. A comparison of the prokaryotic and eukaryotic protein synthesis processes and particularly the observation of the functional and structural similarity between the prokaryotic cell membrane (the interface to the cell exterior) and the membrane of the eukaryotic endoplasmic reticulum (one of the compartments within the endomembrane system) inspire a description that refers to either the eukaryotic endoplasmic reticulum or its membrane or the endomembrane system altogether as a representation of the extracellular medium. However, unless the terms in such a statement are carefully analysed and refined the description would be just a colloquial usage of the concept of “representation” rather than a biosemiotic statement. Another problem associated with employing the concept of “representation” in a biosemiotic context is due to the fact that it may evoke the impression of a conscious mind as the “owner” of the representation. In this paper theories about the emergence of the eukaryotic endomembrane system, as well as its functionality within secretory protein synthesis will be analysed in order to specify in what sense the concept of “representation” can be employed in this context without implying consciousness. Such a study is expected not only to provide an insight into the conditions and assumptions under which this concept can be used for lower level biological processes but also to shed some light on how representations emerge in general.

Opening the SecYEG Protein Translocon

The Sec61/SecYEG translocon is the central component of the major biosynthetic pathway for membrane and secreted proteins. To incorporate membrane proteins into the endoplasmic reticulum membrane of eukaryotes (Sec61), or into the plasma membrane of prokaryotes (SecYEG), the translocon relies on the opening of a lateral helical gate formed by transmembrane (TM) helices TM2 and TM7. The crystallographic structure of the closed state of the translocon (B. van der Berg et al, Nature 427, 36-44, 2003) solved in the absence of bound signal peptide provides valuable information on possible scenarios of membrane protein insertion. Nevertheless, the sequence of structural rearrangements that leads to the opening of the translocon and the geometry of the translocon in its open state remain unknown. To derive information on the structure and dynamics of possible open conformations of the SecYEG translocon, we performed prolonged molecular dynamics simulations of the SecYEG translocon in which we mutated residues determined to participate in stabilizing interactions of the closed state. We have also performed a simulation of the SecYEG translocon with a bound signal peptide. The computations provide insights into interactions essential for keeping the lateral gate closed and how perturbations of these interactions cause rearrangements of the gate.This work was supported by research grants from the National Institute for General Medical Sciences, the NIH National Center for Research Resources, and the National Science Foundation.

Role of Protein Translocation Pathways across the Endoplasmic Reticulum in Trypanosoma brucei

The translocation of secretory and membrane proteins across the endoplasmic reticulum (ER) membrane is mediated by co-translational (via the signal recognition particle (SRP)) and post-translational mechanisms. In this study, we investigated the relative contributions of these two pathways in trypanosomes. A homologue of SEC71, which functions in the post-translocation chaperone pathway in yeast, was identified and silenced by RNA interference. This factor is essential for parasite viability. In SEC71-silenced cells, signal peptide (SP)-containing proteins traversed the ER, but several were mislocalized, whereas polytopic membrane protein biogenesis was unaffected. Surprisingly trypanosomes can interchangeably utilize two of the pathways to translocate SP-containing proteins except for glycosylphosphatidylinositol-anchored proteins, whose level was reduced in SEC71-silenced cells but not in cells depleted for SRP68, an SRP-binding protein. Entry of SP-containing proteins to the ER was significantly blocked only in cells co-silenced for the two translocation pathways (SEC71 and SRP68). SEC63, a factor essential for both translocation pathways in yeast, was identified and silenced by RNA interference. SEC63 silencing affected entry to the ER of both SP-containing proteins and polytopic membrane proteins, suggesting that, as in yeast, this factor is essential for both translocation pathways in vivo. This study suggests that, unlike bacteria or other eukaryotes, trypanosomes are generally promiscuous in their choice of mechanism for translocating SP-containing proteins to the ER, although the SRP-independent pathway is favored for glycosylphosphatidylinositol-anchored proteins, which are the most abundant surface proteins in these parasites.

A role for the two-helix finger of the SecA ATPase in protein translocation.

An important step in the biosynthesis of many proteins is their partial or complete translocation across the plasma membrane in prokaryotes or the endoplasmic reticulum membrane in eukaryotes 1. In bacteria, secretory proteins are generally translocated after completion of their synthesis by the interplay of the cytoplasmic ATPase SecA and a protein-conducting channel formed by the SecY complex 2. How SecA moves substrates through the SecY channel is unclear. However, a recent structure of a SecA-SecY complex raises the possibility that the polypeptide chain is moved by a two-helix finger domain of SecA that is inserted into the cytoplasmic opening of the SecY channel 3. Here, we have used disulfide-bridge crosslinking to show that the loop at the tip of the two-helix finger interacts with a polypeptide chain right at the entrance into the SecY pore. Mutagenesis demonstrates that a tyrosine in the loop is particularly important for translocation, but can be replaced by some other bulky, hydrophobic residues. We propose that the two-helix finger of SecA moves a polypeptide chain into the SecY channel with the tyrosine providing the major contact with the substrate, a mechanism analogous to that suggested for hexameric, protein-translocating ATPases.

Coq2p function in the Saccharomyces cerevisiae coenzyme Q biosynthetic pathway

Respiratory electron transport in the plasma membrane of prokaryotes and in the inner mitochondrial membrane of eukaryotes is supported by the crucial lipid component, ubiquinone (coenzyme Q or Q). Here we further characterize the Coq2 polypeptide, a key resident polypeptide of a complex of Coq polypeptides that is necessary to manufacture Q. One mutant allele of Coq2p and a hemaglutanin tagged Coq2p are described and compared to the wild‐type polypeptide in S. cerevisiae via Western blotting, growth curves and lipid analyses. Although stable levels of Coq2 polypeptide are evident in mitochondria isolated from the coq2‐1 mutant, the strain is incapable of aerobic respiration. We also present biochemical evidence that Coq2p is expressed in two forms, one at 30 kDa and another at 41kDa. Growth on non‐fermentable carbon sources invokes the 41 kDa isoform. We speculate that the 41kDa form is necessary for Q synthesis since it is lacking in the coq2‐1 mutant, and not highly expressed in cell extracts of cells grown in media containing fermentable sugars. We describe the coenzyme Q intermediates present in lipid extracts of the coq2‐1mutant. Our data demonstrate that Coq2p is essential for Q synthesis.This work was supported by NIH grants GM 45952 and NIA AG 19777.

Acrobatic flexibility of the translocon

The machinery that inserts membrane proteins into the ER is unexpectedly flexible, say Kida et al. Even with several segments of a multispanning membrane protein already looped around inside the pore, more can be inserted. Figure 1 A trapped translocating intermediate (left) fits two hydrophilic segments (gray lines) within a single translocon pore. Upon releasing the trap (green), translocation proceeds normally (right). Proteins are inserted into the ER in eukaryotes and the plasma membrane in prokaryotes by the Sec61-based translocon. Data from several studies, including crystal structures, indicate that the translocon's pore consists of a single Sec61α subunit, thus creating a narrow channel. But the new studies suggest the pore is much larger than expected. The authors trapped intermediates in the translocation process by adding a streptavidin-binding peptide tag to the NH2-terminal end of the inserted protein. When streptavidin was added, translocation stalled, resulting in intermediates. The addition of biotin removed the streptavidin and revived translocation. The intermediates revealed that stalled translocating peptides within the pore do not jam the translocon. Two distant hydrophilic segments of the same protein fit within the pore at the same time. As each of these lipid-averse segments needs Sec61α to protect it from the membranous environment, the structure suggests that the pore is not as narrow as previously thought. Another intermediate revealed that a stalled hydrophilic domain did not prevent the subsequent membrane insertion of as many as six successive hydrophobic segments. To explain the flexibility, the authors suggest that perhaps two Sec61 complexes—one for each hydrophilic segment—combine for a translocation event. This model is supported by previous EM images of Sec61 oligomers. It is also possible that a single Sec61 complex undergoes drastic, unpredicted conformational changes or is somehow made larger by accessory proteins. Reference: Kida, Y., et al. 2007. J. Cell Biol. 179:1441–1452. [PubMed]

Ribosome Binding of a Single Copy of the SecY Complex: Implications for Protein Translocation

The SecY complex associates with the ribosome to form a protein translocation channel in the bacterial plasma membrane. We have used cryo-electron microscopy and quantitative mass spectrometry to show that a nontranslating E. coli ribosome binds to a single SecY complex. The crystal structure of an archaeal SecY complex was then docked into the electron density maps. In the resulting model, two cytoplasmic loops of SecY extend into the exit tunnel near proteins L23, L29, and L24. The loop between transmembrane helices 8 and 9 interacts with helices H59 and H50 in the large subunit RNA, while the 6/7 loop interacts with H7. We also show that point mutations of basic residues within either loop abolish ribosome binding. We suggest that SecY binds to this primary site on the ribosome and subsequently captures and translocates the nascent chain.

Protein translocation across the eukaryotic endoplasmic reticulum and bacterial plasma membranes

A decisive step in the biosynthesis of many proteins is their partial or complete translocation across the eukaryotic endoplasmic reticulum membrane or the prokaryotic plasma membrane. Most of these proteins are translocated through a protein-conducting channel that is formed by a conserved, heterotrimeric membrane-protein complex, the Sec61 or SecY complex. Depending on channel binding partners, polypeptides are moved by different mechanisms: the polypeptide chain is transferred directly into the channel by the translating ribosome, a ratcheting mechanism is used by the endoplasmic reticulum chaperone BiP, and a pushing mechanism is used by the bacterial ATPase SecA. Structural, genetic and biochemical data show how the channel opens across the membrane, releases hydrophobic segments of membrane proteins laterally into lipid, and maintains the membrane barrier for small molecules.

X-ray Structures of the Signal Recognition Particle Receptor Reveal Targeting Cycle Intermediates

The signal recognition particle (SRP) and its conjugate receptor (SR) mediate cotranslational targeting of a subclass of proteins destined for secretion to the endoplasmic reticulum membrane in eukaryotes or to the plasma membrane in prokaryotes. Conserved active site residues in the GTPase domains of both SRP and SR mediate discrete conformational changes during formation and dissociation of the SRP·SR complex. Here, we describe structures of the prokaryotic SR, FtsY, as an apo protein and in two different complexes with a non-hydrolysable GTP analog (GMPPNP). These structures reveal intermediate conformations of FtsY containing GMPPNP and explain how the conserved active site residues position the nucleotide into a non-catalytic conformation. The basis for the lower specificity of binding of nucleotide in FtsY prior to heterodimerization with the SRP conjugate Ffh is also shown. We propose that these structural changes represent discrete conformational states assumed by FtsY during targeting complex formation and dissociation.

Single molecule studies of molecular diffusion in cellular membranes: Determining membrane structure

Since the advent of single particle/molecule microscopies, researchers have applied these techniques to understanding the fluid membranes of cells. By observing diffusion of membrane proteins and lipids in live cell membranes of eukaryotic cells, it has been found that membranes contain a mosaic of fluid compartments. Such structure may be instrumental in understanding key characteristics of the membrane. Recent single molecule observations on prokaryotic cell membranes will also be discussed.

Characterization of a Divergent Sec61β Gene in Microsporidia

The general secretory (Sec) pathway is the main mechanism for protein secretion and insertion into endoplasmic reticulum and plasma membrane in prokaryotes and eukaryotes. However, the complete genome of the highly specialized microsporidian parasite Encephalitozoon cuniculi appears to lack a gene for Sec61β, one of three universally conserved proteins that form the core of the Sec translocon. We have identified a putative, highly divergent homologue of Sec61β in the genome of another microsporidian, Antonospora locustae, and used this to identify a previously unrecognized Sec61β in E. cuniculi. The identity of these genes is supported by evidence from secondary structure prediction and gene order conservation. Their functional conservation is confirmed by expressing both microsporidian homologues in yeast, where they are localized to the endoplasmic reticulum and rescue a yeast Sec61β deletion mutant.

Comparison of Biophysical and Biologic Properties of α‐Helical Enantiomeric Antimicrobial Peptides

In our previous study (Chen et al. J Biol Chem 2005, 280:12316-12329), we utilized an alpha-helical antimicrobial peptide V(681) as the framework to study the effects of peptide hydrophobicity, amphipathicity, and helicity on biologic activities where we obtained several V(681) analogs with dramatic improvement in peptide therapeutic indices against gram-negative and gram-positive bacteria. In the present study, the D-enantiomers of three peptides--V(681), V13A(D) and V13K(L) were synthesized to compare biophysical and biologic properties with their enantiomeric isomers. Each D-enantiomer was shown by circular dichroism spectroscopy to be a mirror image of the corresponding L-isomer in benign conditions and in the presence of 50% trifluoroethanol. L- and D-enantiomers exhibited equivalent antimicrobial activities against a diverse group of Pseudomonas aeruginosa clinical isolates, various gram-negative and gram-positive bacteria and a fungus. In addition, L- and D-enantiomeric peptides were equally active in their ability to lyse human red blood cells. The similar activity of L- and D-enantiomeric peptides on prokaryotic or eukaryotic cell membranes suggests that there are no chiral receptors and the cell membrane is the sole target for these peptides. Peptide D-V13K(D) showed significant improvements in the therapeutic indices compared with the parent peptide V(681) by 53-fold against P. aeruginosa strains, 80-fold against gram-negative bacteria, 69-fold against gram-positive bacteria, and 33-fold against Candida albicans. The excellent stability of D-enantiomers to trypsin digestion (no proteolysis by trypsin) compared with the rapid breakdown of the L-enantiomers highlights the advantage of the D-enantiomers and their potential as clinical therapeutics.