N-terminal Transmembrane Domain Research Articles

Crystal Structure of the Electron Carrier Domain of the Reaction Center Cytochrome cz Subunit from Green Photosynthetic Bacterium Chlorobium tepidum

In green sulfur photosynthetic bacteria, the cytochrome cz (cyt cz) subunit in the reaction center complex mediates electron transfer mainly from menaquinol/cytochrome c oxidoreductase to the special pair (P840) of the reaction center. The cyt cz subunit consists of an N-terminal transmembrane domain and a C-terminal soluble domain that binds a single heme group. The periplasmic soluble domain has been proposed to be highly mobile and to fluctuate between oxidoreductase and P840 during photosynthetic electron transfer. We have determined the crystal structure of the oxidized form of the C-terminal functional domain of the cyt cz subunit (C-cyt cz) from thermophilic green sulfur bacterium Chlorobium tepidum at 1.3-Å resolution. The overall fold of C-cyt cz consists of four α-helices and is similar to that of class I cytochrome c proteins despite the low similarity in their amino acid sequences. The N-terminal structure of C-cyt cz supports the swinging mechanism previously proposed in relation with electron transfer, and the surface properties provide useful information on possible interaction sites with its electron transfer partners. Several characteristic features are observed for the heme environment: These include orientation of the axial ligands with respect to the heme plane, surface-exposed area of the heme, positions of water molecules, and hydrogen-bond network involving heme propionate groups. These structural features are essential for elucidating the mechanism for regulating the redox state of cyt cz.

Expression and purification of the transmembrane domain of Fukutin-I for biophysical studies

Fukutin-I is a member of a family of putative O-linked glycosyltransferases linked to the glycosylation of the dystrophin complex. Mutations in this family of proteins have been linked to a number of congenital muscular dystrophies that arise from the hypoglycosylation of α-dystroglycan. Critical to the function of Fukutin and other members of this family is their localisation within the cell, which has been shown to depend critically on the interactions between the N-terminal transmembrane domain of these proteins and the lipid bilayer within the ER/Golgi. To investigate how the interactions between the N-terminal transmembrane domain and the lipid bilayer regulate the localisation of Fukutin-I, we have developed an efficient expression and purification protocol in Escherichia coli to allow biophysical studies to be performed. Expressing the N-terminal domain of Fukutin-1 fused to a His6 tag resulted in the localisation of the protein to the bacterial membrane. A purification strategy has been developed to isolate the highly hydrophobic transmembrane domain of Fukutin-1 from the membrane with yields of approximately 4mg per litre of minimal media. Preliminary biophysical analyses have confirmed the identity of the peptide and revealed that in hydrophobic solvents mimicking the bilayer, the peptide adopts a well-structured α-helix as predicted from the sequence.

Role of C2 domain proteins during synaptic vesicle exocytosis

Neurotransmitter release is mediated by the fusion of synaptic vesicles with the presynaptic plasma membrane. Fusion is triggered by a rise in the intracellular calcium concentration and is dependent on the neuronal SNARE (soluble N-ethylmaleimide-sensitive fusion protein-attachment protein receptor) complex. A plethora of molecules such as members of the MUNC13, MUNC18, complexin and synaptotagmin families act along with the SNARE complex to enable calcium-regulated synaptic vesicle exocytosis. The synaptotagmins are localized to synaptic vesicles by an N-terminal transmembrane domain and contain two cytoplasmic C2 domains. Members of the synaptotagmin family are thought to translate the rise in intracellular calcium concentration into synaptic vesicle fusion. The C2 domains of synaptotagmin-1 bind membranes in a calcium-dependent manner and in response induce a high degree of membrane curvature, which is required for its ability to trigger membrane fusion in vitro and in vivo. Furthermore, members of the soluble DOC2 (double-C2 domain) protein family have similar properties. Taken together, these results suggest that C2 domain proteins such as the synaptotagmins and DOC2s promote membrane fusion by the induction of membrane curvature in the vicinity of the SNARE complex. Given the widespread expression of C2 domain proteins in secretory cells, it is proposed that promotion of SNARE-dependent membrane fusion by the induction of membrane curvature is a widespread phenomenon.

A Role for Lipid-Protein Interaction in Intergral Membrane Protein Function

The fukutins are a family of eukaryotic membrane proteins whose mis-localisation within the cell has been shown to be associated with the onset of Fukuyama muscular dystrophy. The interaction between the N-terminal transmembrane domain fukutin with the atypical lipid bilayer of the Golgi apparatus has been postulated to play a crucial role in the retention of the protein there.To investigate the nature of these interactions we have developed a bacterial expression system for isolating the N-terminal transmembrane domain of Fukutin-I. We have characterised this hydrophobic peptide using molecular biology techniques, mass spectrometry, circular dichroism and nuclear magnetic resonance studies.Currently, we are using solid state NMR and fluorescence studies to analyse how the oligomeric state, lateral segregation and structure of fukutin reconstituted into artificial bilayers varies as a function of varying lipid compositions. These studies will provide further insight into the role of lipid-protein interactions in membrane protein structure and assembly, segregation into lipid rafts and the role these properties may play in protein trafficking.

Crystal Structures of Trypanosoma brucei Sterol 14α-Demethylase and Implications for Selective Treatment of Human Infections

Sterol 14alpha-demethylase (14DM, the CYP51 family of cytochrome P450) is an essential enzyme in sterol biosynthesis in eukaryotes. It serves as a major drug target for fungal diseases and can potentially become a target for treatment of human infections with protozoa. Here we present 1.9 A resolution crystal structures of 14DM from the protozoan pathogen Trypanosoma brucei, ligand-free and complexed with a strong chemically selected inhibitor N-1-(2,4-dichlorophenyl)-2-(1H-imidazol-1-yl)ethyl)-4-(5-phenyl-1,3,4-oxadi-azol-2-yl)benzamide that we previously found to produce potent antiparasitic effects in Trypanosomatidae. This is the first structure of a eukaryotic microsomal 14DM that acts on sterol biosynthesis, and it differs profoundly from that of the water-soluble CYP51 family member from Mycobacterium tuberculosis, both in organization of the active site cavity and in the substrate access channel location. Inhibitor binding does not cause large scale conformational rearrangements, yet induces unanticipated local alterations in the active site, including formation of a hydrogen bond network that connects, via the inhibitor amide group fragment, two remote functionally essential protein segments and alters the heme environment. The inhibitor binding mode provides a possible explanation for both its functionally irreversible effect on the enzyme activity and its selectivity toward the 14DM from human pathogens versus the human 14DM ortholog. The structures shed new light on 14DM functional conservation and open an excellent opportunity for directed design of novel antiparasitic drugs.

Impact of Disease-causing SUR1 Mutations on the KATP Channel Subunit Interface Probed with a Rhodamine Protection Assay

The function of the ATP-sensitive potassium (K(ATP)) channel relies on the proper coupling between its two subunits: the pore-forming Kir6.2 and the regulator SUR. The conformation of the interface between these two subunits can be monitored using a rhodamine 123 (Rho) protection assay because Rho blocks Kir6.2 with an efficiency that depends on the relative position of transmembrane domain (TMD) 0 of the associated SUR (Hosy, E., Dérand, R., Revilloud, J., and Vivaudou, M. (2007) J. Physiol. 582, 27-39). Here we find that the natural and synthetic K(ATP) channel activators MgADP, zinc, and SR47063 induced a Rho-insensitive conformation. The activating mutation F132L in SUR1, which causes neonatal diabetes, also rendered the channel resistant to Rho block, suggesting that it stabilized an activated conformation by uncoupling TMD0 from the rest of SUR1. At a nearby residue, the SUR1 mutation E128K impairs trafficking, thereby reducing surface expression and causing hyperinsulinism. To augment channel density at the plasma membrane to investigate the effect of mutating this residue on channel function, we introduced the milder mutation E126A at the matching residue of SUR2A. Mutation E126A imposed a hypersensitive Rho phenotype indicative of a functional uncoupling between TMD0 and Kir6.2. These results suggest that the TMD0-Kir6.2 interface is mobile and that the gating modes of Kir6.2 correlate with distinct positions of TMD0. They further demonstrate that the second intracellular loop of SUR, which contains the two residues studied here, is a key structural element of the TMD0-Kir6.2 interface.

Cloning and molecular characterization of the potato RING finger protein gene StRFP1 and its function in potato broad-spectrum resistance against Phytophthora infestans

Really interesting new gene (RING) finger proteins function as ubiquitin ligase and play key roles in biotic and abiotic stresses. A new RING-H2 finger protein gene, StRFP1, was cloned from Phytophthora infestans-inoculated leaves of potato ( Solanum tuberosum) clone 386209.10, which is free of R1-R11 genes. The deduced amino acid sequence was characterized by an N-terminal transmembrane domain, a GLD region and a RING-H2 finger signature. StRFP1 is homologous to the tobacco NtACRE132 protein and belongs to the ATL family. The DNA gel blot analysis and mapping revealed that StRFP1, an intron-free gene, had one to two copies in the potato genome and was located on chromosome 3. RT-PCR assays showed that StRFP1 was constitutively expressed in potato plants and significantly induced in detached potato leaves by P. infestans and plant defense-related signal molecules, abscisic acid, salicylic acid and methyl jasmonate. Transient expression studies revealed that StRFP1 fused with GFP localized to the plasma membrane or out of that in onion epidermal cells. The function of StRFP1 in potato resistance against late blight was further investigated by constructing overexpression and RNA interference (RNAi) vectors, which were introduced into potato cv. E-potato 3, respectively. By challenging the detached leaves with mixture races of P. infestans, all of the StRFP1-overexpressing plants displayed slower disease development than non-transformed controls in terms of the lesion growth rate (LGR). In contrast, StRFP1-silencing plants through RNAi were more susceptible to pathogen infection. The present results demonstrate that StRFP1 contributes to broad-spectrum resistance against P. infestans in potato.

FdTonB is involved in the photoregulation of cellular morphology during complementary chromatic adaptation in Fremyella diplosiphon

We have characterized a Fremyella diplosiphon TonB protein (FdTonB) and investigated its function during complementary chromatic adaptation. Sequence similarity analysis of FdTonB (571 aa) led to identification of several conserved domains characteristic of TonB proteins, including an N-terminal transmembrane domain, a central proline-rich spacer and a C-terminal TonB-related domain (TBRD). We identified a novel glycine-rich domain containing (Gly-X)(n) repeats. To assess FdTonB function, we constructed a DeltatonB mutant through homologous recombination based upon truncation of the central proline-rich spacer, glycine-rich domain and TBRD. Our DeltatonB mutant exhibited an aberrant cellular morphology under green light, with expanded cell width compared to the parental wild-type (WT) strain. The cellular morphology of the DeltatonB mutant recovered upon WT tonB expression. Interestingly, tonB expression was found to be independent of RcaE. As DeltatonB and WT strains respond in the same way when grown under iron-replete versus iron-limited conditions, our results suggest that FdTonB is not involved in the classic TonB function of mediating cellular adaptation to iron limitation, but exhibits a novel function related to the photoregulation of cellular morphology in F. diplosiphon.

Tuning the Cellular Trafficking of the Lysosomal Peptide Transporter TAPL by its N-terminal Domain

The homodimeric ATP-binding cassette (ABC) transport complex TAPL (transporter associated with antigen processing-like, ABCB9) translocates a broad spectrum of peptides from the cytosol into the lumen of lysosomes. The presence of an extra N-terminal transmembrane domain (TMD0) lacking any sequence homology to known proteins distinguishes TAPL from most other ABC transporters of its subfamily. By dissecting TAPL, we could assign distinct functions to the core complex and TMD0. The core-TAPL complex, composed of six predicted transmembrane helices and a nucleotide-binding domain, is sufficient for peptide transport, showing that the core transport complex is correctly targeted to and assembled in the membrane. Strikingly, in contrast to the full-length transporter, the core translocation complex is targeted preferentially to the plasma membrane. However, TMD0 alone, comprising a putative four transmembrane helix bundle, traffics to lysosomes. Upon coexpression, TMD0 forms a stable non-covalently linked complex with the core translocation machinery and guides core-TAPL into lysosomal compartments. Therefore, TMD0 represents a unique domain, which folds independently and encodes the information for lysosomal targeting. These outcomes are discussed in respect of trafficking, folding and function of TAPL.

The N-Terminal Transmembrane Domain of λ S Is Required for Holin but Not Antiholin Function

The lambda S gene encodes a holin, S105, and an antiholin, S107, which differs by its Met-Lys N-terminal extension. The model for the lysis-defective character of S107 stipulates that the additional N-terminal basic residue keeps S107 from assuming the topology of S105, which is N-out, C-in, with three transmembrane domains (TMDs). Here we show that the N terminus of S105 retains its fMet residue but that the N terminus of S107 is fully deformylated. This supports the model that in S105, TMD1 inserts into the membrane very rapidly but that in S107, it is retained in the cytoplasm. Further, it reveals that, compared to S105, S107 has two extra positively charged moieties, Lys2 and the free N-terminal amino group, to hinder its penetration into an energized membrane. Moreover, an allele, S105(DeltaTMD1), with TMD1 deleted, was found to be defective in lysis, insensitive to membrane depolarization, and dominant to the wild-type allele, indicating that the lysis-defective, antiholin character of S107 is due to the absence of TMD1 from the bilayer rather than to its ectopic localization at the inner face of the cytoplasmic membrane. Finally, the antiholin function of the deletion protein was compromised by the substitution of early-lysis missense mutations in either the deletion protein or parental S105 but restored when both S105(DeltaTMD1) and holin carried the substitution.

Overexpression, characterization, and crystallization of the functional domain of cytochrome c z from Chlorobium tepidum

Cytochrome c(z) is found in green sulfur photosynthetic bacteria, and is considered to be the only electron donor to the special pair P840 of the reaction center. It consists of an N-terminal transmembrane domain and a C-terminal soluble domain that binds a single heme group. Large scale expression of the C-terminal functional domain of the cytochrome c(z) (C-cyt c(z)) from the thermophilic bacterium Chlorobium tepidum has been achieved using the Escherichia coli expression system. The C-cyt c(z) expressed has been highly purified, and is stable at room temperature over 10 days of incubation for both reduced and oxidized forms. Spectroscopic measurements indicate that the heme iron in C-cyt c(z) is in a low-spin state and this does not change with the redox state. (1)H-NMR spectra of the oxidized C-cyt c(z) exhibited unusually large paramagnetic chemical shifts for the heme methyl protons in comparison with those of other Class I ferric cytochromes c. Differences in the (1)H-NMR linewidth were observed for some resonances, indicating different dynamic environments for these protons. Crystals of the oxidized C-cyt c(z) were obtained using ammonium sulfate as a precipitant. The crystals diffracted X-rays to a maximum resolution of 1.2 A, and the diffraction data were collected to 1.3 A resolution.

Role of N-glycosylation of the SEA module of rodent Muc3 in posttranslational processing of its carboxy-terminal domain

A prominent feature of the rodent Muc3 SEA module is the precursor cleavage event that segregates the O-glycosylated N-terminal fragment and transmembrane domain into the noncovalently attached heterodimer. There are seven potential N-glycosylation sites that occur in a cluster in the SEA module of Muc3. However, it is unknown if these sites are modified or what the function of these N-glycans may be in the SEA module. Our data show that the proteolytic cleavage of the rodent Muc3 SEA module was partially prevented by treatment with tunicamycin, an inhibitor of N-glycosylation. Each single mutant of the seven N-glycosylation sites (N1A, N2A, N3A, N4A, N5A, N6A, and N7A) and multiple mutants, including double (N34A) and triple (N345A) mutants, and mutants with four (N3457A), five (N34567A), six (N134567A and N234567A), seven (N1234567A) mutations, confirmed that all seven of these potential sites are N-glycosylated simultaneously. The proteolytic cleavage of the SEA module was not affected when it lacked only one, two, or three N-glycans, but was partially inhibited when lacking four, five, and six N-glycans. In all, 2%, 48%, 85%, and 73% of the products from N3457A, N34567A, N134567A, and N234567A transfectants, respectively, remained uncleaved. The proteolytic cleavage was completely prevented in the N1234567A transfectant, which eliminated all seven N-glycans in the SEA module. The interaction of the heterodimer was independent of the N-glycans within the rodent Muc3 SEA module. Thus, the N-glycosylation pattern constituted a control point for the modulation of the proteolytic cleavage of the SEA module.

Pleiotropic phenotypes caused by an opal nonsense mutation in an essential gene encoding HMG‐CoA reductase in fission yeast

Schizosaccharomyces pombe genome contains an essential gene hmg1(+) encoding the sterol biosynthetic enzyme, 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR). Here, we isolated an allele of the hmg1(+) gene, hmg1-1/its12, as a mutant that showed sensitivities to high temperature and to FK506, a calcineurin inhibitor. The hmg1-1 allele contained an opal nonsense mutation in its N-terminal transmembrane domain, yet in spite of the mutation a full-length protein was produced, suggesting a read-through termination codon. Consistently, overexpression of the hmg1-1 mutant gene suppressed the mutant phenotypes. The hmg1-1 mutant showed hypersensitivity to pravastatin, an HMGR inhibitor, suggesting a defective HMGR activity. The mutant treated with FK506 caused dramatic morphological changes and showed defects in cell wall integrity, as well as displayed synthetic growth phenotypes with the mutant alleles of genes involved in cytokinesis and cell wall integrity. The mutant exhibited different phenotypes from those of the disruption mutants of ergosterol biosynthesis genes, and it showed normal filipin staining as well as showed normal subcellular localization of small GTPases. These data suggest that the pleiotropic phenotypes reflect the integrated effects of the reduced availability of ergosterol and various intermediates of the mevalonate pathway.

GmRFP1 encodes a previously unknown RING-type E3 ubiquitin ligase in Soybean (Glycine max)

RING-finger proteins with E3 ubiquitin ligase activity play important roles in the regulation of plant growth and development. In this study, a cDNA clone encoding a novel RING-finger protein, designated as GmRFP1, was isolated and characterized from soybean. GmRFP1 was an intronless gene encoding a predicted protein product of 392 amino acid residues with a molecular mass of ~43 kDa. The protein contained a RING-H2 motif and an N-terminal transmembrane domain. The transcript was observed in all detected organs and was up-regulated by abscisic acid (ABA) and salt stress, but down-regulated by cold and drought treatments. We further expressed and purified both wild type and mutant version of GmRFP1 in E. coli. In vitro assays showed that the purified GmRFP1 induced the formation of polyubiquitin chains while mutation within the RING-finger region abolished the ubiquitination activity. These findings suggest that GmRFP1 is a previously unknown E3 ubiquitin ligase in soybean and that the RING domain is required for its activity. It may play unappreciated roles in ABA signaling and stress responses via mediating the ubiquitination and degradation of target proteins through the ubiquitin-proteasome pathway.

Cleavage and Secretion of Epstein-Barr Virus Glycoprotein 42 Promote Membrane Fusion with B Lymphocytes

Epstein-Barr virus (EBV) membrane glycoprotein 42 (gp42) is required for viral entry into B lymphocytes through binding to human leukocyte antigen (HLA) class II on the B-cell surface. EBV gp42 plays multiple roles during infection, including acting as a coreceptor for viral entry into B cells, binding to EBV glycoprotein H (gH) and gL during the process of membrane fusion, and blocking T-cell recognition of HLA class II-peptide complexes through steric hindrance. EBV gp42 occurs in two forms in infected cells, a full-length membrane-bound form and a soluble form generated by proteolytic cleavage that is secreted from infected cells due to loss of the N-terminal transmembrane domain. Both the full-length and the secreted gp42 forms bind to gH/gL and HLA class II, and the functional significance of gp42 cleavage is currently unclear. We found that in a virus-free cell-cell fusion assay, enhanced secretion of gp42 promoted fusion with B lymphocytes, and mutation of the site of gp42 cleavage inhibited membrane fusion activity. The site of gp42 cleavage was found to be physically distinct from the residues of gp42 necessary for binding to gH/gL. These results suggest that cleavage and secretion of gp42 are necessary for the process of membrane fusion with B lymphocytes, providing the first indicated functional difference between full-length and cleaved, secreted gp42.

Distinct Transport and Intracellular Activities of Two GlcAT-P Isoforms

A neural glycotope, human natural killer-1 carbohydrate, is involved in synaptic plasticity. The key biosynthetic enzyme is a glucuronyltransferase, GlcAT-P, a type II membrane protein comprising an N-terminal cytoplasmic tail, transmembrane domain, stem region, and C-terminal catalytic domain. Previously, we reported that GlcAT-P has two isoforms differing in only the presence or absence of the N-terminal 13 amino acids (P-N13) in the cytoplasmic tail, but the functional distinction of these two isoforms has not been reported. Herein, we show that when expressed in Neuro2A cells, short form GlcAT-P (sGlcAT-P) exhibited significantly higher glucuronylation activity than the longer form (lGlcAT-P), despite their comparable specific activities in vitro. In addition, sGlcAT-P was strictly localized in Golgi apparatus, whereas lGlcAT-P was mainly localized in Golgi but partly in the endoplasmic reticulum. We demonstrated that the small GTPase, Sar1, recognized a dibasic motif in the cytoplasmic tail near P-N13 that was important for exiting the endoplasmic reticulum, and Sar1 interacted with sGlcAT-P more strongly than lGlcAT-P. Finally, the attachment of P-N13 to another glycosyltransferase, polysialyltransferase-I (ST8Sia-IV), had similar effects, such as reduced activity and entrapment within endoplasmic reticulum. These results suggest that P-N13 can control glycosyltransferase transport through Sar1 binding interference.

Chimeric hepatitis B and C viruses envelope proteins can form subviral particles: implications for the design of new vaccine strategies

The hepatitis B virus (HBV) envelope protein (S) self-assembles into subviral particles used as commercial vaccines against hepatitis B. These particles are excellent carriers for foreign epitopes, which can be inserted into the external hydrophilic loop or at the N- or C-terminal end of the HBV S protein. We show here that the N-terminal transmembrane domain (TMD) of HBV S can be replaced by the TMDs of the hepatitis C virus (HCV) envelope proteins E1 and E2, to generate fusion proteins containing the entire HCV E1 or E2 sequence that are efficiently coassembled with the HBV S into particles. This demonstrates the remarkable tolerance of the HBV S protein to sequence substitutions conserving its subviral particle assembly properties. These findings may have implications for the design of new vaccine strategies based on the use of HBV subviral particles as carriers for various transmembrane proteins and produced using the same industrial procedures that are established for the HBV vaccine.

Combined use of mass spectrometry and heterologous expression for identification of membrane-interacting peptides in cytochrome P450 46A1 and NADPH-cytochrome P450 oxidoreductase

Cytochrome P450 46A1 (CYP46A1) and NADPH-cytochrome P450 oxidoreductase (CPR) are the components of the brain microsomal mixed-function monooxygenase system that catalyzes the conversion of cholesterol to 24-hydroxycholesterol. Both CYP46A1 and CPR are monotopic membrane proteins that are anchored to the endoplasmic reticulum via the N-terminal transmembrane domain. The exact mode of peripheral association of CYP46A1 and CPR with the membrane is unknown. Therefore, we studied their membrane topology by using an approach in which solution-exposed portion of heterologously expressed membrane-bound CYP46A1 or CPR was removed by digestion with either trypsin or chymotrypsin followed by extraction of the residual peptides and their identification by mass spectrometry. The identified putative membrane-interacting peptides were mapped onto available crystal structures of CYP46A1 and CPR and the proteins were positioned in the membrane considering spatial location of the missed cleavage sites located within these peptide as well as the flanking residues whose cleavage produced these peptides. Experiments were then carried out to validate the inference from our studies that the substrate, cholesterol, enters CYP46A1 from the membrane. As for CPR, its putative membrane topology indicates that the Q153R and R316W missense mutations found in patients with disordered steroidogenesis are located within the membrane-associated regions. This information may provide insight in the deleterious nature of these mutations.

Multiple Kv Channel-interacting Proteins Contain an N-terminal Transmembrane Domain That Regulates Kv4 Channel Trafficking and Gating

Kv channel-interacting proteins (KChIPs) are auxiliary subunits of the heteromultimeric channel complexes that underlie neuronal I(SA), the subthreshold transient K(+) current that dynamically regulates membrane excitability, action potential firing properties, and long term potentiation. KChIPs form cytoplasmic associations with the principal pore-forming Kv4 subunits and typically mediate enhanced surface expression and accelerated recovery from depolarization-induced inactivation. An exception is KChIP4a, which dramatically suppresses Kv4 inactivation while promoting neither surface expression nor recovery. These unusual properties are attributed to the effects of a K channel inactivation suppressor domain (KISD) encoded within the variable N terminus of KChIP4a. Here, we have functionally and biochemically characterized two brain KChIP isoforms, KChIP2x and KChIP3x (also known as KChIP3b) and show that they also contain a functional KISD. Like KChIP4a and in contrast with non-KISD-containing KChIPs, both KChIP2x and KChIP3x strongly suppress inactivation and slow activation and inhibit the typical increases in surface expression of Kv4.2 channels. We then examined the properties of the KISD to determine potential mechanisms for its action. Subcellular fractionation shows that KChIP4a, KChIP2x, and KChIP3x are highly associated with the membrane fraction. Fluorescent confocal imaging of enhanced green fluorescent proteins (eGFP) N-terminally fused with KISD in HEK293T cells indicates that KISDs of KChIP4a, KChIP2x, and KChIP3x all autonomously target eGFP to intracellular membranes. Cell surface biotinylation experiments on KChIP4a indicate that the N terminus is exposed extracellularly, consistent with a transmembrane KISD. In summary, KChIP4a, KChIP2x, and KChIP3x comprise a novel class of KChIP isoforms characterized by an unusual transmembrane domain at their N termini that modulates Kv4 channel gating and trafficking.

Structure of the Type III Secretion Recognition Protein YscU from Yersinia enterocolitica

The inner-membrane protein YscU has an important role during the assembly of the Yersinia enterocolitica type III secretion injectisome. Its cytoplasmic domain (YscU C) recognizes translocators as individual substrates in the export hierarchy. Activation of YscU entails autocleavage at a conserved NPTH motif. Modification of this motif markedly changes the properties of YscU, including translocator export cessation and production of longer injectisome needles. We determined the crystal structures of the uncleaved variants N263A and N263D of YscU C at 2.05 Å and 1.55 Å resolution, respectively. The globular domain is found to consist of a central, mixed β-sheet surrounded by α-helices. The NPTH motif forms a type II β-turn connecting two β-strands. NMR analysis of cleaved and uncleaved YscU C indicates that the global structure of the protein is retained in cleaved YscU C. The structure of YscU C variant N263D reveals that wild type YscU C is poised for cleavage due to an optimal reaction geometry for nucleophilic attack of the scissile bond by the side chain of Asn263. In vivo analysis of N263Q and H266A/R314A YscU variants showed a phenotype that combines the absence of translocator secretion with normal needle-length control. Comparing the structure of YscU to those of related proteins reveals that the linker domain between the N-terminal transmembrane domain and the autocleavage domain can switch from an extended to a largely α-helical conformation, allowing for optimal positioning of the autocleavage domain during injectisome assembly.