C-terminal Transmembrane Segment Research Articles

The periplasmic coiled coil formed by the assembly platform proteins PulL and PulM is critical for function of the Klebsiella type II secretion system

Bacteria use type II secretion systems (T2SS) to secrete to their surface folded proteins that confer diverse functions, from nutrient acquisition to virulence. In the Klebsiella species, T2SS-mediated secretion of pullulanase (PulA) requires assembly of a dynamic filament called the endopilus. The inner membrane assembly platform (AP) subcomplex is essential for endopilus assembly and PulA secretion. AP components PulL and PulM interact with each other through their C-terminal globular domains and transmembrane segments. Here, we investigated the roles of their periplasmic helices, predicted to form a coiled coil, in assembly and function of the PulL–PulM complex. PulL and PulM variants lacking these periplasmic helices were defective for interaction in the bacterial two-hybrid (BACTH) assay. Their functions in PulA secretion and assembly of PulG subunits into endopilus filaments were strongly reduced. Interestingly, deleting the cytoplasmic peptide of PulM nearly abolished the function of variant PulMΔN and its interaction with PulG, but not with PulL, in the BACTH assay. Nevertheless, PulL was specifically proteolyzed in the presence of the PulMΔN variant, suggesting that PulM N-terminal peptide stabilizes PulL in the cytoplasm. We discuss the implications of these results for the T2S endopilus and type IV pilus assembly mechanisms.

Capture and delivery of tail-anchored proteins to the endoplasmic reticulum.

Tail-anchored (TA) proteins fulfill diverse cellular functions within different organellar membranes. Their characteristic C-terminal transmembrane segment renders TA proteins inherently prone to aggregation and necessitates their posttranslational targeting. The guided entry of TA proteins (GET in yeast)/transmembrane recognition complex (TRC in humans) pathway represents a major route for TA proteins to the endoplasmic reticulum (ER). Here, we review important new insights into the capture of nascent TA proteins at the ribosome by the GET pathway pretargeting complex and the mechanism of their delivery into the ER membrane by the GET receptor insertase. Interestingly, several alternative routes by which TA proteins can be targeted to the ER have emerged, raising intriguing questions about how selectivity is achieved during TA protein capture. Furthermore, mistargeting of TA proteins is a fundamental cellular problem, and we discuss the recently discovered quality control machineries in the ER and outer mitochondrial membrane for displacing mislocalized TA proteins.

Molecular Logic of Prokaryotic Surface Layer Structures.

Most prokaryotic cells are encased in a surface layer (S-layer) consisting of a paracrystalline array of repeating lattice-forming proteins. S-layer proteins populate a vast and diverse sequence space, performing disparate functions in prokaryotic cells, including cellular defense, cell-shape maintenance, and regulation of import and export of materials. This article highlights recent advances in the understanding of S-layer structure and assembly, made possible by rapidly evolving structural and cell biology methods. We underscore shared assembly principles revealed by recent work and discuss a common molecular framework that may be used to understand the structural organization of S-layer proteins across bacteria and archaea.

Both positional and chemical variables control in vitro proteolytic cleavage of a presenilin ortholog

Mechanistic details of intramembrane aspartyl protease (IAP) chemistry, which is central to many biological and pathogenic processes, remain largely obscure. Here, we investigated the in vitro kinetics of a microbial intramembrane aspartyl protease (mIAP) fortuitously acting on the renin substrate angiotensinogen and the C-terminal transmembrane segment of amyloid precursor protein (C100), which is cleaved by the presenilin subunit of γ-secretase, an Alzheimer disease (AD)-associated IAP. mIAP variants with substitutions in active-site and putative substrate-gating residues generally exhibit impaired, but not abolished, activity toward angiotensinogen and retain the predominant cleavage site (His-Thr). The aromatic ring, but not the hydroxyl substituent, within Tyr of the catalytic Tyr-Asp (YD) motif plays a catalytic role, and the hydrolysis reaction incorporates bulk water as in soluble aspartyl proteases. mIAP hydrolyzes the transmembrane region of C100 at two major presenilin cleavage sites, one corresponding to the AD-associated Aβ42 peptide (Ala-Thr) and the other to the non-pathogenic Aβ48 (Thr-Leu). For the former site, we observed more favorable kinetics in lipid bilayer-mimicking bicelles than in detergent solution, indicating that substrate-lipid and substrate-enzyme interactions both contribute to catalytic rates. High-resolution MS analyses across four substrates support a preference for threonine at the scissile bond. However, results from threonine-scanning mutagenesis of angiotensinogen demonstrate a competing positional preference for cleavage. Our results indicate that IAP cleavage is controlled by both positional and chemical factors, opening up new avenues for selective IAP inhibition for therapeutic interventions.

Acetylation is essential for nuclear heme oxygenase-1-enhanced tumor growth and invasiveness.

Overexpression of heme oxygenase-1 (HO-1), an endoplasmic reticulum-anchored enzyme, is observed in many cancers. HO-1 nuclear translocation has been shown to correlate with progression of several cancers. We recently reported that HO-1 is susceptible to intramembrane proteolysis and translocates to the nucleus to promote cancer growth and invasiveness without depending on its enzymatic activity. In the present study, we show that the HO-1 lacking C-terminal transmembrane segment (t-HO-1) was susceptible to acetylation by p300 and CREB-binding protein (CBP) histone acetyltransferase in the nucleus. Mass spectrometry analysis of HO-1 isolated from human embryonic kidney cells 293T (HEK293T) cells overexpressing CBP and t-HO-1 revealed two acetylation sites located at K243 and K256. Mutation of both lysine residues to arginine (R) abolished t-HO-1-enhanced tumor cell growth, migration and invasion. However, mutation of the lysine residues to glutamine (Q), a mimic of acetylated lysine, had no significant effect on t-HO-1-mediated tumorigenicity. Mechanistic studies demonstrated that transcriptional factor JunD interacted with wild-type (WT) t-HO-1 and mutant carrying K243/256Q but not K243/256 R mutation. Moreover, JunD-induced AP-1 transcriptional activity was significantly enhanced by coexpression with WT and acetylation-mimic but not acetylation-defective t-HO-1. Consistent with the in vitro observations, the implication of K243/256 acetylation in t-HO-1-enhanced tumorigenicity was also demonstrated in xenograft models. Immunohistochemistry performed with a specific antibody against acetyl-HO-1 showed the positive acetyl-HO-1 nuclear staining in human lung cancer tissues but not in the corresponding non-tumor tissues, supporting its clinical significance. Collectively, our findings highlight the importance of nuclear HO-1 post-translational modification in the induction of cancer progression.

439 - The Nuclear Form of Heme Oxygenase (HO)-1 Preferentially Promotes Glycolysis in Hyperoxia But Fails to Protect Against Heme Toxicity

Heme oxygenase-1 (HO-1) is a stress-inducible enzyme that catalyzes the degradation of heme, generating carbon monoxide and antioxidant bile pigments. Cytoplasmic HO-1 is tethered to the smooth endoplasmic reticulum via a C-terminal trans-membrane segment. Cleavage of this anchor allows for localization of a truncated, catalytically inactive form of HO-1 to the nucleus, as has been observed in fetal lung cells in hyperoxia and in several cancer tissues. While cytoplasmic HO-1 is associated with cytoprotection directly via its antioxidant activities, nuclear HO-1 also plays a role in protection against oxidative injury via its interaction with other nuclear proteins and transcription factors, resulting in altered gene expression of several rate-limiting enzymes in key metabolic pathways including glycolysis. Interestingly, nuclear HO-1 is also associated with a proliferative phenotype. Although HO-1 has been shown to play a role in glucose metabolism and altered mitochondrial dynamics, the mechanism by which this occurs is not known. In an effort to investigate this, wild type and HO-1-/- mouse embryonic fibroblast (MEF) cells as well as MEF expressing only the nuclear, truncated HO (HO-TR), were evaluated for glycolysis and oxidative phosphorylation (oxphos) after hyperoxic exposure (95% O2/5% CO2 for 24 hours) and controls were exposed to air/5% CO2 for 24 hours. The HO-TR had increased glycolytic activity compared to HO+/+ or HO-/- cells. In contrast, oxphos, in particular spare respiratory capacity, was no different between any of the groups in hyperoxia. However, the presence of excess heme (0-50 micromolar) inhibited oxphos activity in the MEF lacking HO-1 catalytic activity, namely, HO-TR and HO-/-, in a dose dependent fashion. Our results suggest a role of nuclear HO-1 in enhancing glycolysis during hyperoxic stress thereby protecting against injury and promoting cell proliferation. They also suggest that catalytically active HO-1 is required for homeostasis of mitochondial function. Taken together our results suggest that nuclear HO-1 is cytoprotective under hyperoxic stress via a metabolic shift toward glycolysis and highlight a putative role of HO-1 in mitochondrial dynamics and metabolic regulation.

The Human Sodium-Glucose Cotransporter (hSGLT1) Is a Disulfide-Bridged Homodimer with a Re-Entrant C-Terminal Loop.

Na-coupled cotransporters are proteins that use the trans-membrane electrochemical gradient of Na to activate the transport of a second solute. The sodium-glucose cotransporter 1 (SGLT1) constitutes a well-studied prototype of this transport mechanism but essential molecular characteristics, namely its quaternary structure and the exact arrangement of the C-terminal transmembrane segments, are still debated. After expression in Xenopus oocytes, human SGLT1 molecules (hSGLT1) were labelled on an externally accessible cysteine residue with a thiol-reactive fluorophore (tetramethylrhodamine-C5-maleimide, TMR). Addition of dipicrylamine (DPA, a negatively-charged amphiphatic fluorescence “quencher”) to the fluorescently-labelled oocytes is used to quench the fluorescence originating from hSGLT1 in a voltage-dependent manner. Using this arrangement with a cysteine residue introduced at position 624 in the loop between transmembrane segments 12 and 13, the voltage-dependent fluorescence signal clearly indicated that this portion of the 12–13 loop is located on the external side of the membrane. As the 12–13 loop begins on the intracellular side of the membrane, this suggests that the 12–13 loop is re-entrant. Using fluorescence resonance energy transfer (FRET), we observed that different hSGLT1 molecules are within molecular distances from each other suggesting a multimeric complex arrangement. In agreement with this conclusion, a western blot analysis showed that hSGLT1 migrates as either a monomer or a dimer in reducing and non-reducing conditions, respectively. A systematic mutational study of endogenous cysteine residues in hSGLT1 showed that a disulfide bridge is formed between the C355 residues of two neighbouring hSGLT1 molecules. It is concluded that, 1) hSGLT1 is expressed as a disulfide bridged homodimer via C355 and that 2) a portion of the intracellular 12–13 loop is re-entrant and readily accessible from the extracellular milieu.

Permuting the PGF Signature Motif Blocks both Archaeosortase-Dependent C-Terminal Cleavage and Prenyl Lipid Attachment for the Haloferax volcanii S-Layer Glycoprotein.

For years, the S-layer glycoprotein (SLG), the sole component of many archaeal cell walls, was thought to be anchored to the cell surface by a C-terminal transmembrane segment. Recently, however, we demonstrated that the Haloferax volcanii SLG C terminus is removed by an archaeosortase (ArtA), a novel peptidase. SLG, which was previously shown to be lipid modified, contains a C-terminal tripartite structure, including a highly conserved proline-glycine-phenylalanine (PGF) motif. Here, we demonstrate that ArtA does not process an SLG variant where the PGF motif is replaced with a PFG motif (slg(G796F,F797G)). Furthermore, using radiolabeling, we show that SLG lipid modification requires the PGF motif and is ArtA dependent, lending confirmation to the use of a novel C-terminal lipid-mediated protein-anchoring mechanism by prokaryotes. Similar to the case for the ΔartA strain, the growth, cellular morphology, and cell wall of the slg(G796F,F797G) strain, in which modifications of additional H. volcanii ArtA substrates should not be altered, are adversely affected, demonstrating the importance of these posttranslational SLG modifications. Our data suggest that ArtA is either directly or indirectly involved in a novel proteolysis-coupled, covalent lipid-mediated anchoring mechanism. Given that archaeosortase homologs are encoded by a broad range of prokaryotes, it is likely that this anchoring mechanism is widely conserved. Prokaryotic proteins bound to cell surfaces through intercalation, covalent attachment, or protein-protein interactions play critical roles in essential cellular processes. Unfortunately, the molecular mechanisms that anchor proteins to archaeal cell surfaces remain poorly characterized. Here, using the archaeon H. volcanii as a model system, we report the first in vivo studies of a novel protein-anchoring pathway involving lipid modification of a peptidase-processed C terminus. Our findings not only yield important insights into poorly understood aspects of archaeal biology but also have important implications for key bacterial species, including those of the human microbiome. Additionally, insights may facilitate industrial applications, given that photosynthetic cyanobacteria encode uncharacterized homologs of this evolutionarily conserved enzyme, or may spur development of unique drug delivery systems.

Structural characterization of Get3/Get4/Get5 involved in TA-protein targeting

During the biosynthesis of tail-anchored (TA) membrane proteins, their single C-terminal trans-membrane segment is inserted into the ER membrane for orientating the functional domain(s) towards the cytosolic side of the cell. The machinery responsible for this post-translational process has only recently come to light. In yeast, the proteins participating in TA protein insertion include Get1/Get2, Get3, Get4 and Get5. Although structural information and the individual roles of most components of this system have been defined, the interactions and interplay between them remain to be elucidated. Here, we investigated the interactions between Get3 and the Get4/Get5 complex (Get4/5) from Saccharomyces cerevisiae. We show that Get3 interacts with Get4/5 via an interface dominated by electrostatic forces. Using isothermal titration calorimetry and small-angle X-ray scattering, we further demonstrate that the Get3 homodimer interacts with two copies of the Get4/5 complex to form an extended conformation in solution.

The topology of the C-terminal sections of the NCX1 Na+/Ca2+ exchanger and the NCKX2 Na+/Ca2+-K+ exchanger

Mammalian Na+/Ca2+ (NCX) and Na+/Ca2+-K+ exchangers (NCKX) are polytopic membrane proteins that play critical roles in calcium homeostasis in many cells. Although hydropathy plots for NCX and NCKX are very similar, reported topological models for NCX1 and NCKX2 differ in the orientation of the three C-terminal transmembrane segments (TMS). NCX1 is thought to have 9 TMS and a re-entrant loop, whereas NCKX2 is thought to have 10 TMS. The current topological model of NCKX2 is very similar to the 10 membrane spanning helices seen in the recently reported crystal structure of NCX_MJ, a distantly related archaebacterial Na+/Ca2+ exchanger. Here we reinvestigate the orientation of the three C-terminal TMS of NCX1 and NCKX2 using mass-tagging experiments of substituted cysteine residues. Our results suggest that NCX1, NCKX2 and NCX_MJ all share the same 10 TMS topology.

The Open Conformation of a Voltage-Gated Sodium Channel Reveals the Transmembrane Pathway and Gating Mechanism

In excitable cells, the initiation of an action potential results from the opening of voltage-gated sodium channels. In humans, mutations in different sodium channel isoforms have been shown to have causal relationships with neurological and cardiovascular diseases, and they therefore represent key targets for development of pharmaceutical drugs. Sodium channels are also present in some prokaryotes, where they appear to function in homeostasis, motility, and chemotaxis. All sodium channels undergo a series of conformational changes associated with their open, closed and inactivated functional states.We have determined the crystal structure of the open conformation of the NavMs bacterial sodium channel pore (McCusker et al, 2012). It contrasts with the structure (Payandeh et al, 2011) of a closely-related bacterial channel, NavAb, which has a closed pore conformation. The differences, which in the open form produce an internal cavity accessible to the cytoplasmic surface, result from a bend/rotation about a central residue in the C-terminal transmembrane segment. The open activation gate is of sufficient diameter to allow passage of hydrated sodium ions. Comparisons between the forms suggest a mechanism of channel opening and closing which differs both from that of potassium channels, and from existing models for sodium channel opening/closing. Comparisons of the selectivity filters and fenestrations further suggest features important both for activity and for the design of new state-specific ligands/drugs.

SnapShot: Viral and Eukaryotic Protein Fusogens

Viral Fusion Effectors Viral fusogens are anchored in the viral membrane, and the initial fusogenic conformational change allows them to insert into the cell membrane. This transition is triggered by interactions of the virus particle with the host cell—either binding a cell surface receptor or protons in the acidic endosomal environment. During the fusogenic conformational change, the fusion protein initially adopts an intermediate extended conformation in which a “fusion peptide” projects away from the virus particle and inserts into the target membrane. The fusion protein subsequently collapses into a “hairpin” conformation in which the fusion peptide becomes juxtaposed to the viral transmembrane (TM) segment, thereby juxtaposing the two membranes. The structures of the postfusion hairpin conformation for a number of viruses are presented, highlighting common structural features of the observed hairpins. The overall process of virus-cell fusion and the accompanying conformational changes of the effector proteins have been reviewed, see for example Harrison (2008). The viral fusion proteins analyzed so far fall into three structural classes in which the postfusion form is a trimer of hairpins with different architectures. The predicted location of the fused membrane is outlined above the trimers. The class I motif is characterized by a 7 residue periodicity of nonpolar amino acids (i.e.,“heptad repeats”) that give rise to a central parallel trimeric a-helical coiled coil along the long axis of a rod-shaped molecule (blue in the ribbon diagrams). The N and C termini (labeled in blue and cyan) pack against each other at the membrane proximal end. A “stutter,” a region where the coiled helix unwinds and alters the nonpolar repeat pattern (in red), permits the structural alignment of the various hairpin trimers (Igonet et al., 2011). The heptad repeats are also interrupted by hydrophilic layers (indicated in magenta, with horizontal arrows). Class II proteins are folded essentially as b sheets with no long coiled a helix, and the trimer of hairpins is formed by the parallel interactions of domains I and II (red and yellow). The trimer is further clamped in place by the lateral interactions of domain III (in blue), which stabilizes the hairpin conformation. A stem region (only partially present in the available structures) connects to the TM segment. The fusion peptide is internal, situated in the loop between two b strands, and is therefore termed “fusion loop” (FL). Class III proteins appear to be intermediate between classes I and II: they have a central parallel coiled coil (blue), but they have an elongated, b-sheet-rich fusion domain N terminal to it (yellow) instead of a fusion peptide as in class I. Although this fusion domain is superficially similar to its counterpart in class II proteins, the topological b-strand organization is unrelated, suggesting convergent rather than divergent evolution. The central parallel coiled coil also has a stutter, allowing a close alignment to the class I fusion proteins. The fusion domain of class III proteins has two fusion loops labeled FL1 and FL2. Although there is no amino acid sequence conservation across virus families, the structures show clear homology within classes II and III. However, the simple heptad repeat pattern of class I fusogens is in several cases not sufficient to distinguish between divergent or convergent evolution and therefore, only for a subset of families displayed in the figure do the fusion proteins appear as clearly homologous. Fusion of Intracellular Vesicles A variety of different proteins control the fusion of intracellular vesicles, but the actual fusion effectors are believed to be the SNARE proteins, as reviewed by Sudhof and Rothman (2009). The prefusion form of the SNAREs appears to be largely unstructured, and the postfusion form consists of a hetero-oligomeric complex formed by SNARE subunits that were anchored in the two opposing membranes before their fusion. The SNAREs in their postfusion conformation share with viral proteins of classes I and III an elongated, a-helical, parallel oligomeric coiled-coil organization in which the C-terminal TM segments are juxtaposed at the same end, making a stable protein rod termed “SNAREpin” by analogy to the viral hairpin. The SNAREpin organization differs from the viral proteins because it is a four-stranded coiled coil in which the amino acid sequence of each a helix is different. At the center, there is a hydrophilic, or “ionic,” layer consisting of a central arginine (R) and three glutamine (Q) residues that hydrogen bond the guanidinium group of the arginine. This feature has allowed the classification of the SNAREs into four different categories—R-SNAREs and Qa-, Qb-, and Qc-SNAREs—with one representative of each being necessary for formation of a functional SNAREpin, as displayed in the figure.

Microscopic Analysis of Severe Structural Rearrangements of the Plant Endoplasmic Reticulum and Golgi Caused by Overexpression ofPoa semilatent virusMovement Protein

Cell-to-cell transport of plant viruses is mediated by virus-encoded movement proteins and occurs through plasmodesmata interconnecting neighboring cells in plant tissues. Three movement proteins coded by the “triple gene block” (TGB) and named TGBp1, TGBp2 and TGBp3 have distinct functions in viral transport. TGBp1 binds viral genomic RNAs to form ribonucleoprotein complexes representing the transport form of viral genome, while TGBp2 and TGBp3 are necessary for intracellular delivery of such complexes to plasmodesmata. Recently, it was revealed that overexpression of Potato virus X TGBp3 triggers the unfolded protein response mitigating the endoplasmic reticulum (ER) stress leading to cell death if this protein reaches high levels in the ER. Here we report microscopic studies of the influence of the Poa semilatent hordeivirus TGBp3 overexpressed in Nicotiana benthamiana epidermal cells by particle bombardment on cell endomembranes and demonstrate that the protein C-terminal transmembrane segment contains a determinant responsible for vesiculation and coalescence of the endoplasmic reticulum and Golgi presumably accompanying the ER stress that can be induced upon high-level TGBp3 expression.

Mitochondrion-dependent N-terminal Processing of Outer Membrane Mcl-1 Protein Removes an Essential Mule/Lasu1 Protein-binding Site

Mcl-1, a pro-survival member of the Bcl-2 family located at the mitochondrial outer membrane, is subject to constitutive ubiquitylation by the Bcl-2 homology 3-only E3 ligase, Mule/Lasu1, resulting in rapid steady-state degradation via the proteasome. Insertion of newly synthesized Mcl-1 into the mitochondrial outer membrane is dependent on its C-terminal transmembrane segment, but once inserted, the N terminus of a portion of the Mcl-1 molecules can be subject to proteolytic processing. Remarkably, this processing requires an intact electrochemical potential across the inner membrane. Three lines of evidence directed at the endogenous protein, however, indicate that the resulting Mcl-1ΔN isoform resides in the outer membrane: (i) full-length Mcl-1 and Mcl-1ΔN resist extraction by alkali but are accessible to exogenous protease; (ii) almost the entire populations of Mcl-1 and Mcl-1ΔN are accessible to the membrane-impermeant Cys-reactive agent 4-acetamido-4'-[(iodoacetyl)amino]stilbene-2,2'-disulfonic acid; and (iii) Mcl-1 and Mcl-1ΔN exhibit equivalent chemical cross-linking to Bak in intact mitochondria, an Mcl-1 binding partner located in the outer membrane. In addition to the Mule Bcl-2 homology 3 domain, we show that interaction between Mcl-1 and Mule also requires the extreme N terminus of Mcl-1, which is lacking in Mcl-1ΔN. Thus, Mcl-1ΔN does not interact with Mule, exhibits reduced steady-state ubiquitylation, evades the hyper-rapid steady-state degradation that is observed for full-length Mcl-1 in response to treatments that limit global protein synthesis, and confers resistance to UV stress-induced cell death.

Cooperative and independent activities of Sgt2 and Get5 in the targeting of tail-anchored proteins

Abstract TPR proteins modulate the activity of molecular chaperones. Here, we describe the S. cerevisiae TPR protein Sgt2 as interaction partner of Ssa1 and Hsp104 and as a component of the GET pathway by interacting with Get5. The GET pathway mediates the sorting of tail-anchored (TA) proteins, harboring a C-terminal trans-membrane segment, to the ER membrane. S. cerevisiae sgt2Δ cells show partial defects in TA protein sorting. Sgt2 activity in vivo relies on its N- and C-terminal domains, whereas the central TPR domain and thus chaperone interactions are dispensable. We show that TA protein sorting defects are more severe in sgt2Δ get5Δ mutants compared to single knockouts. Furthermore, overproduction of Sgt2 becomes toxic to get3Δ but not to get5Δ cells. Together, these findings indicate an additional, Get5-independent role of Sgt2 in TA protein sorting, pointing to parallel pathways of substrate delivery to Get3.

Formation of protein complexes containing plant virus movement protein TGBp3 is necessary for its intracellular trafficking

Cell-to-cell movement of Poa semilatent virus (genus Hordeivirus) in infected plants is mediated by three viral ‘triple gene block’ (TGB) proteins. One of those termed TGBp3 is an integral membrane protein essential for intracellular transport of other TGB proteins and viral genomic RNA to plasmodesmata. TGBp3 targeting to plasmodesmata-associated sites is believed to involve an unconventional mechanism which does not employ endoplasmic reticulum-derived transport vesicles. Previously TGBp3 has been shown to contain a composite transport signal consisting of the central hydrophilic protein region which includes a conserved pentapeptide YQDLN and the C-terminal transmembrane segment. This study demonstrates that these TGBp3 structural elements have distinct functions in protein transport. The YQDLN-containing region is essential for TGBp3 incorporation into high-molecular-mass protein complexes. In transient expression assay formation of such complexes is necessary for entering the TGBp3-specific pathway of intracellular transport and protein delivery to plasmodesmata-associated sites. In virus-infected plants TGBp3 is also found predominantly in the form of high-molecular-mass complexes. When the complex-formation function of YQDLN-containing region is disabled by a mutation, targeting to plasmodesmata-associated sites can be complemented by a heterologous peptide capable of formation multimeric complexes. The C-terminal transmembrane segment is found to be an essential signal of TGBp3 intracellular transport to peripheral sites.

Structural and Functional Characterization of the C-terminal Transmembrane Region of NBCe1-A

NBCe1-A and AE1 both belong to the SLC4 HCO(3)(-) transporter family. The two transporters share 40% sequence homology in the C-terminal transmembrane region. In this study, we performed extensive substituted cysteine-scanning mutagenesis analysis of the C-terminal region of NBCe1-A covering amino acids Ala(800)-Lys(967). Location of the introduced cysteines was determined by whole cell labeling with a membrane-permeant biotin maleimide and a membrane-impermeant 2-((5(6)-tetramethylrhodamine)carboxylamino) ethyl methanethiosulfonate (MTS-TAMRA) cysteine-reactive reagent. The results show that the extracellular surface of the NBCe1-A C-terminal transmembrane region is minimally exposed to aqueous media with Met(858) accessible to both biotin maleimide and TAMRA and Thr(926)-Ala(929) only to TAMRA labeling. The intracellular surface contains a highly exposed (Met(813)-Gly(828)) region and a cryptic (Met(887)-Arg(904)) connecting loop. The lipid/aqueous interface of the last transmembrane segment is at Asp(960). Our data clearly determined that the C terminus of NBCe1-A contains 5 transmembrane segments with greater average size compared with AE1. Functional assays revealed only two residues in the region of Pro(868)-Leu(967) (a functionally important region in AE1) that are highly sensitive to cysteine substitution. Our findings suggest that the C-terminal transmembrane region of NBCe1-A is tightly folded with unique structural and functional features that differ from AE1.

1H, 13C and 15N resonance assignments of the rhodanese domain of YgaP from Escherichia coli

Rhodanese domain is a ubiquitous structural module commonly found in bacterial, archaeal and eukaryotic cells. Growing evidence indicates that rhodanese domains act as the carrier of reactive sulfur atoms by forming persulfide intermediates in distinct metabolic pathways. YgaP, a membrane protein consisting of a rhodanese domain and a C-terminal transmembrane segment, is the only membrane-associated rhodanese in Escherichia coli. Herein, we report the resonance assignments of (1)H, (13)C and (15)N atoms of rhodanese domain of YgaP. Totally, chemical shifts of more than 95% of the atoms were assigned.

Conserved Negative Charges in the Transmembrane Segments of Subunit K of the NADH:Ubiquinone Oxidoreductase Determine Its Dependence on YidC for Membrane Insertion

All members of the Oxa1/Alb3/YidC family have been implicated in the biogenesis of respiratory and energy transducing proteins. In Escherichia coli, YidC functions together with and independently of the Sec system. Although the range of proteins shown to be dependent on YidC continues to increase, the exact role of YidC in insertion remains enigmatic. Here we show that YidC is essential for the insertion of subunit K of the NADH:ubiquinone oxidoreductase and that the dependence is due to the presence of two conserved glutamate residues in the transmembrane segments of subunit K. The results suggest a model in which YidC serves as a membrane chaperone for the insertion of the less hydrophobic, negatively charged transmembrane segments of NuoK.

A Universal Zone in the Ribosomal Exit Tunnel for Helix Formation in Kv1.3

Crystal structures of Kv channels have given us a reasonably complete view of the structure of a mature Kv channel. However, details of its structure acquisition are missing. We now report on the biogenesis of secondary structure of the transmembrane segments and intervening linkers of Kv1.3. Using a combination of accessibility assays, both cysteine pegylation (Tu et al., 2007; Lu and Deutsch, 2005) and N-linked glycosylation (Mingarro et al., 2000), we derive the following principles of folding for nascent sequences within their native contexts. First, native helical transmembrane sequences initially form helices only within the distal 20Å of the ribosomal tunnel near the exit port. We refer to this region as the α-zone. Helix formation in transmembrane segments thus occurs vectorially from N- to C-terminus as each segment moves sequentially into the α-zone. Second, linker sequences also form compact structures inside the α-zone even in the absence of helix formation of their C-terminal flanking transmembrane segments. Third, helical structures, whether transmembrane or linker segments, formed in the tunnel retain their helicity in the translocon. These principles emerge from a diversity of native transmembrane and linker sequences that comprise Kv1.3 and may therefore be applicable to protein biogenesis in general. [Supported by NIH grant GM 52302].