No description available.

Most membrane proteins are inserted into the membrane co-translationally utilizing the translocon, which allows a sufficiently long and hydrophobic stretch of amino acids to partition into the membrane. However, X-ray structures of membrane proteins have revealed that some transmembrane helices (TMHs) are surprisingly hydrophilic. These marginally hydrophobic transmembrane helices (mTMH) are not recognized as TMHs by the translocon in the absence of local sequence context. We have studied three native mTMHs, which were previously shown to depend on a subsequent TMH for membrane insertion. Their recognition was not due to specific interactions. Instead, the presence of basic amino acids in their cytoplasmic loop allowed membrane insertion of one of them. In the other two, basic residues are not sufficient unless followed by another, hydrophobic TMH. Post-insertional repositioning are another way to bring hydrophilic residues into the membrane. We show how four long TMHs with hydrophilic residues seen in X-ray structures, are initially inserted as much shorter membrane-embedded segments. Tilting is thus induced after membrane-insertion, probably through tertiary packing interactions within the protein. Aquaporin 1 illustrates how a mTMH can shape membrane protein folding and how repositioning can be important in post-insertional folding. It initially adopts a four-helical intermediate, where mTMH2 and TMH4 are not inserted into the membrane. Consequently, TMH3 is inserted in an inverted orientation. The final conformation with six TMHs is formed by TMH2 and 4 entering the membrane and TMH3 rotating 180°. Based on experimental and computational results, we propose a mechanism for the initial step in the folding of AQP1: A shift of TMH3 out from membrane core allows the preceding regions to enter the membrane, which provides flexibility for TMH3 to re-insert in its correct orientation. / <p>At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 2: Manuscript.</p>

The expression of polysaccharide capsules is common in bacteria and associated with virulence in some pathogenic strains. Strains of the Gram-negative bacterium Escherichia coli express a structurally diverse range of capsular polysaccharides. E. coli strains expressing group 2 capsules are associated with a number of extra-intestinal infections, including sepsis, urinary tract infections, and neonatal meningitis. Group 2 capsular polysaccharides are synthesised on the cytoplasmic face of the inner membrane. Evidence from previous work suggests that export of polysaccharides across the Gram-negative membranes involves four transport proteins which interact to form a continuous membrane-spanning translocation complex (the KpsMTED translocon). Polysaccharide translocation across the inner membrane requires the ABC transporter KpsMT, in which KpsM is the integral inner membrane component and KpsT is the ATPase. Transport across the periplasmic space and outer membrane involves the integral inner membrane protein KpsE and the outer membrane protein KpsD, respectively. This thesis addressed some of the key areas in the study of group 2 polysaccharide transport by employing the K5 capsule as a model system. Using biochemical and molecular genetics approaches, the study focused on establishing functional and structural characteristics of the translocon members and analysing protein-protein interactions within the complex. This study demonstrated that KpsE can self-associate as dimers, tetramers and possibly higher order oligomers in the absence of other capsule gene products and the K5 substrate. A mutagenesis study of KpsE revealed that the periplasmic, membrane-associated C-terminus is essential for correct protein function. Work presented here confirmed previous data, which suggested a direct interaction between KpsE and KpsM, by alternative methods, and demonstrated that the C-terminal domain of KpsE is required for this interaction. Further experiments suggested that KpsE and KpsM can both form higher order oligomers when interacting as a complex. The C-terminus of KpsE is not required for an interaction between KpsE and KpsD, and the two proteins are thus more likely to interact via their respective periplasmic domains. Generation of a theoretical model of the secondary structure and topology of KpsD predicted that KpsD is made primarily of β-sheets with some interspersed α-helices, including a larger coiled coil region. The theoretical topology model proposed an N-terminal transmembrane domain made of eight membrane-spanning regions, and a large periplasmic domain. Substituted-cysteine accessibility method and myc-epitope insertion analysis were both assessed for their suitability for topology analysis of KpsD. Myc-epitope insertion was identified as the recommended approach for future topology study. Myc-epitope tagging of the periplasmic C-terminus of KpsD revealed that a native C-terminus is essential for correct KpsD function.In conclusion, this thesis contributes to the model of group 2 polysaccharide export in E. coli, and, more generally, provides clues about the transport of high-molecular weight molecules across Gram-negative membranes. It is hoped that a thorough understanding of polysaccharide transport might reveal therapeutic targets to block capsule export in pathogenic E. coli in the future.

The Type III Secretion (T3S) system is a system utilized by many pathogenic bacteria to inject proteins into host cells during an infection. Effector proteins enter the host cell by passing through the proteinaceous T3S translocon, which forms a pore on the host cell membrane. Pseudomonas aeruginosa is an opportunistic pathogen that utilizes the T3S system, and very little is known about how the P. aeruginosa translocon forms. The proteins PopB and PopD are believed to assemble into the P. aeruginosa translocon. A pore-forming heterocomplex of PopB and PopD has been reconstituted in model membranes, however this heterocomplex has not been assessed in its relation to the translocon formed on the host cell. The interaction of this heterocomplex with other T3S system components was measured to determine if this complex acts similarly to the translocon. Initial assays that can be used to compare the molecular weight of the translocon isolated from eukaryotic cells after P. aeruginosa contact to the calculated molecular weight of the heterocomplex were developed as well. This study provides insight into how the PopB:PopD heterocomplex formed in model membranes relates to the translocon formed during a P. aeruginosa infection.

[ES] Los virus de plantas son los agentes causales de un gran número de enfermedades en plantas que ocasionan grandes pérdidas económicas. El virus de las manchas necróticas del melón (MNSV), es un pequeño virus de RNA monocatenario de polaridad positiva, perteneciente al género Gammacarmovirus, cuyo genoma codifica cinco proteínas. La proteína de cubierta (CP), está formada por tres dominios distintos. El descubrimiento de un péptido de transito dual en la región amino-terminal de la CP fue el punto de partida de esta tesis. Al inicio de una infección por MNSV, la CP nuevamente sintetizada es transportada al interior de cloroplastos y mitocondrias mientras que, una parte mucho menor se mantiene en el citoplasma aumentando a medida que avanza la infección. La inhibición de este transporte dual conlleva un aumento de la actividad supresora del silenciamiento del RNA de la CP. Sin embargo, la infección sistémica se ve particularmente afectada. Por tanto, la acumulación de la CP en el citoplasma puede provocar un aumento de la replicación viral pero a su vez una sobreexpresión de la p29, puede provocar una explosión oxidativa y una necrosis que restringe el movimiento viral. De este modo, el transporte de la CP a los orgánulos podría evitar una replicación viral excesiva mediante la modulación de la actividad supresora para gestionar el equilibrio entre la defensa de la planta y la contradefensa viral favoreciendo una interacción compatible entre ambos. Desafortunadamente, Arabidopsis thaliana no es huésped para el MNSV. Por tanto, para entender mejor el transporte de la CP a estos orgánulos, se identificaron los receptores y los poros de los translocones de las membranas externas de las mitocondrias y los cloroplastos en Nicotiana benthamiana. Esta caracterización funcional se realizó principalmente mediante VIGS y RT-qPCR, que mostró una redundancia funcional mayor que la observada entre los homólogos de Arabidopsis. Además, esta herramienta también se utilizó para evaluar la relevancia de cada componente bajo la infección por MNSV, y junto con los estudios de interacción CP-receptor realizados mediante BiFC y Y2H, nos permitió identificar NbToc159A para cloroplastos y NbOm64 para mitocondrias, como principales receptores. A su vez, el silenciamiento de NbToc34, NbToc75 o NbTom40 resultó en una resistencia generalizada no solo a MNSV sino también al virus del arrugamiento del nabo (TCV) y al virus del moteado del clavel (CarMV), lo que respalda la idea actualmente aceptada y que involucra el estado fisiológico del cloroplasto y la mitocondria en la señalización temprana de la respuesta defensiva. Finalmente, se realizó una búsqueda de factores del huésped que interaccionasen con la CP mediante con TurboID, una ligasa de biotina, que permite la detección de interacciones tanto directas e indirectas como transitorias y estables. Así, se obtuvo un gran número de proteínas candidatas utilizando la CP de MNSV y su mutante de localización citoplásmica, ∆NtCP. Tres de ellas, NbSIK1, NbSMU2 y NbMAP3K mostraron un efecto perjudicial constante y repetitivo sobre la acumulación del RNA viral. Después de la validación de las interacciones mediante otro método, y el análisis de la localización subcelular de la CP bajo el silenciamiento del interactor correspondiente, se establecieron dos hipótesis principales. En primer lugar, dado que la función principal de NbSMU2 está relacionada con el procesamiento y regulación del RNA mensajero, esta proteína podría ser secuestrada por la CP provocando la expresión de genes provirales. Por otro lado, NbSIK1 y NbMAP3K, actúan como reguladores positivo y negativo de la respuesta PTI a la infección, respectivamente. Además, ambas proteínas interaccionan entre sí y forman parte de la cascada de MAP quinasas, por lo que en nuestra segunda hipótesis, la CP interaccionaría con este complejo, promoviendo una regulación negativa de la PTI que facilitaría el desarrollo de la infección. / [CA] Els virus de plantes són els principals causants de la major part de malalties en plantes i les consegüents pèrdues econòmiques. El virus de les taques necròtiques del meló (MNSV) és un virus menut d'RNA monocatenari de polaritat positiva, pertanyent al gènere Gammacarmovirus, el genoma del qual codifica cinc proteïnes. La proteïna de coberta (CP) està formada per tres dominis diferents. El descobriment d'un pèptid de trànsit dual a la part aminoterminal de la CP va ser el punt de partida d'aquesta tesi. A l'inici d'una infecció per MNSV, la CP novament sintetitzada és transportada a l'interior dels cloroplasts i mitocondris mentre que, una part molt menor es manté al citoplasma augmentant a mesura que avança la infecció. La inhibició d'aquest transport dual comporta un augment de l'activitat supressora del silenciament de l'RNA de la CP. No obstant això, la infecció sistèmica es va frenar. Per tant, l'acumulació de la CP al citoplasma pot provocar un augment de la replicació viral però alhora una sobreexpressió de la p29, una replicasa auxiliar que ocasiona alteracions morfològiques als mitocondris, una explosió oxidativa i necrosi que restringeix el moviment viral. D'aquesta manera, el transport de la CP als orgànuls podria evitar una replicació viral excessiva mitjançant la modulació de l'activitat supressora per gestionar l'equilibri entre defensa de la planta i contradefensa viral que condueixen a una interacció compatible entre tots dos. Per entendre millor el mecanisme molecular que regeix el transport de la CP a aquests orgànuls, es van identificar els receptors i els porus dels translocon de les membranes externes dels mitocondris i els cloroplasts a Nicotiana benthamiana. Aquesta caracterització funcional es va realitzar principalment mitjançant VIGS i RT-qPCR, que va mostrar una redundància funcional més gran que l'observada entre els homòlegs d'Arabidopsis. A més, aquesta eina també es va utilitzar per avaluar la rellevància de cada component sota la infecció per MNSV, i juntament amb els estudis d'interacció CP-receptor realitzats mitjançant BiFC i Y2H, ens va permetre identificar a NbToc159A per a cloroplasts i NbOm64 per a mitocondris, com els principals receptors implicats en el transport de la CP a aquests orgànuls. Alhora, el silenciament de NbToc34, NbToc75 o NbTom40 va resultar en una resistència generalitzada a MNSV, TCV i CarMV, la qual cosa recolza la idea que circula actualment i que involucra l'estat fisiològic del cloroplast i el mitocondri en la senyalització primerenca de la resposta defensiva. Finalment, es va fer una cerca de factors de l'hoste que interaccionessin amb la CP mitjançant la innovadora tècnica de marcatge de proximitat amb TurboID, una lligasa de biotina, que permet la detecció d'interaccions tant directes i indirectes com transitòries i estables. Així, es va obtenir un gran nombre de proteïnes candidates utilitzant la CP de MNSV i el seu mutant de localització citoplàsmica, ∆NtCP. Tres de elles, NbSIK1, NbSMU2 i NbMAP3K van mostrar un efecte perjudicial constant i repetitiu sobre l'acumulació de l'RNA viral. Després de la validació de les interaccions mitjançant un altre mètode, i l'examen de la localització subcel·lular de la CP sota el silenciament de cada interactor, es van establir dues hipòtesis principals. En primer lloc, atès que la funció principal de NbSMU2 està relacionada amb el processament i la regulació de l'RNA missatger, aquesta proteïna podria ser segrestada per la CP provocant l'expressió de gens provirals. D'altra banda, NbSIK1 i NbMAP3K actuen com a reguladors positiu i negatiu de la resposta PTI a la infecció, respectivament. A més, les dos proteïnes interaccionen entre si i formen part de la cascada de MAP quinases, per la qual cosa en la nostra segona hipòtesi, la CP interaccionaria amb aquest complex, promovent una regulació negativa de la PTI que facilitaria el desenvolupament de la infecció. / [EN] Plant viruses are the causal agents of many plant diseases and the subsequent economic losses, estimated to be US$60 billion worldwide each year. The melon necrotic spot virus (MNSV) is a small, single-stranded, positive-sense RNA virus that belongs to the genus Gammacarmovirus and encodes five proteins. The coat protein (CP) is composed of three distinct domains. The discovery of a dual transit peptide in the amino-terminal part of the CP was the starting point of this thesis. Early in MNSV infection, the new synthesized CP is imported into chloroplasts and mitochondria, while the cytoplasmic pool increases as the infection progresses. Inhibiting this dual transport leads to an increase in the RNA silencing suppressor activity of the CP. However, far from resulting in an enhanced infection development, systemic spread was impaired. Therefore, the accumulation of cytoplasmic CP may cause an increase in viral replication and overexpression of p29, an auxiliary replicase that causes morphological alterations, ROS, and necrosis that may restrict viral movement. Thus, a new role for CP targeting would be to avoid excessive viral replication by modulating the suppressor activity to manage the balance between plant defense and viral counter-defense, leading to a compatible interaction. Unfortunately, Arabidopsis thaliana is not a host for MNSV. Thus, to better understand the molecular mechanism behind the CP dual targeting, the receptors and pores of the Nicotiana benthamiana mitochondrial and chloroplast outer membrane translocons were genome identified, and some functional characterization was carried out. We assigned the following names NbToc75-III, NbToc34, NbToc90, NbToc120, NbToc159A, NbToc159B, NbTic22-III for chloroplast translocon components, and NbTom40, NbTom20-1, NbTom20-2, NbOm64 for mitochondrion translocon components. The functional characterization was mainly carried out by virus-induced gene silencing (VIGS) and RT-qPCR, revealing a functional redundancy higher than that reported for Arabidopsis homologs. Additionally, VIGS was also used to evaluate the relevance of each translocon component in MNSV infection, and together with CP-receptor interaction studies performed by BiFC and Y2H, allowed us to identify NbToc159A for chloroplasts and NbOm64 for mitochondria as the main receptors involved in the CP organelle import. Moreover, silencing of NbToc34, NbToc75, or NbTom40 resulted in a generalized resistance not only to MNSV but also to turnip crinkle virus (TCV), and carnation mottle virus (CarMV), supporting the current idea that involves the chloroplast and mitochondrion physiological state in early defense response signaling. Finally, a search for host factors interacting with the CP was performed by the innovative TurboID proximity labeling tool, which allows the detection of both direct/indirect and transient/stable interactions. Thus, a large number of candidate proteins were obtained that interacted either with the MNSV CP or with ∆NtCP, a cytoplasm-localized mutant. Three of them, NbSIK1, NbSMU2, and NbMAP3K, showed a consistent and repetitive detrimental effect on MNSV RNA accumulation. After the validation of the interactions using another method and the analysis of the subcellular localization of the MNSV CP under each interactor silencing, two main hypotheses were proposed. Firstly, since the main function of NbSMU2 is related to messenger RNA regulation by splicing, this protein could be sequestered by the CP, causing the expression of proviral genes. On the other hand, NbSIK1 and NbMAP3K act as positive and negative regulators of the PTI response to infection, respectively. Moreover, both proteins interact with each other and are part of the MAP kinase cascade, so in our second hypothesis, CP would interact with this complex, promoting a negative regulation of PTI that would facilitate viral infection. / La autora ha disfrutado de un contrato predoctoral de formación de personal investigador (FPI) (PRE-2018-84130) otorgado por el Ministerio de Ciencia e Innovación asociado al proyecto BIO2017-88321-R. Este trabajo de tesis doctoral ha sido realizado con el apoyo económico de los proyectos de investigación del Ministerio de Ciencia e Innovación, BIO2017-88321-R y PID2020-115571RB- I00. / Sáiz Bonilla, M. (2023). Deciphering the intracellular dual targeting of the melon necrotic spot virus coat protein, its interaction with host factors and their roles in plant defense [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/195836

La sécrétion des protéines est un processus essentiel à la vie. Chez les eucaryotes, les protéines sécrétées transitent dans le réticulum endoplasmique par le pore de translocation. Le translocon est composé de trois sous-unités fondamentales nommées Sec61α, β et γ chez les mammifères, ou Sec61p, Sbh1p et Sss1p chez les levures. Tandis que le rôle des sous-unités α et γ est bien connu, celui de la sous-unité β demeure énigmatique. Plusieurs phénotypes distincts sont associés à cette protéine dans différents organismes, mais le haut niveau de conservation de séquence suggère plutôt une fonction universelle conservée. Récemment, Feng et al. (2007) ont montré que le domaine transmembranaire (TMD) de Sbh1p était suffisant pour complémenter plusieurs phénotypes associés à la délétion du gène chez Saccharomyces cerevisiae, suggérant un rôle important de cette région. L’objectif de mon projet de recherche consiste à étudier la fonction biologique de la sous-unité β du translocon et de son TMD chez Schizosaccharomyces pombe. Dans cette levure, j’ai découvert que le gène sbh1+ n’était pas essentiel à la viabilité à 30oC, mais qu’il était requis pour la croissance à basse température. La délétion de sbh1+ entraîne une sensibilité aux stress de la paroi cellulaire et une diminution de la sécrétion des protéines à 23oC. La surexpression de Sbh1p diminue elle aussi la sécrétion des protéines et altère la morphologie cellulaire. Ces phénotypes sont distincts de ceux observés chez S. cerevisiae, où la délétion des deux paralogues de Sec61β entraîne une sensibilité à haute température plutôt qu’à basse température. Malgré cela, les homologues de Sec61β de S. pombe et de S. cerevisiae sont tout deux capables de complémenter la thermosensibilité respective de chaque levure. La complémentation est possible même avec l’homologue humain de Sec61β, indiquant la conservation d’une fonction de Sec61β de la levure à l’homme. Remarquablement, le TMD de Sec61β de S. pombe, de S. cerevisiae et de l’humain sont suffisants pour complémenter la délétion génomique autant chez la levure à fission que chez la levure à bourgeons. Globalement, ces observations indiquent que le TMD de Sec61β exerce une fonction cellulaire conservée à travers les espèces. / Protein secretion is an essential biological process. In eukaryotes, secreted proteins transit into the endoplasmic reticulum through the translocon pore. The core of the translocation channel is composed of three subunits called Sec61α, β and γ in mammals, or Sec61p, Sbh1p and Sss1p in yeasts. While the role of the α and γ subunit is well understood, the function of the β subunit remains ill-defined. Although numerous species-specific phenotypes have been reported for this protein, the striking sequence conservation among species argue in favour of a universal role. Recently, Feng et al. (2007) reported the surprising finding that the transmembrane domain (TMD) of Sbh1p was sufficient to complement different functions of the entire protein in Saccharomyces cerevisiae, suggesting an important role for this region. The aim of my project was to explore the biological function of the translocon β subunit and its TMD in Schizosaccharomyces pombe. In this yeast, we found that the sbh1+ gene is unessential for viability at 30oC, but is required for growth at low temperature. Knockout of sbh1+ results in sensitivity to cell-wall stress and reduced protein secretion at 23oC. Overexpression of Sbh1p also diminishes protein secretion and results in an elongated cell shape. These phenotypes contrast with those observed S. cerevisiae, as deletion of both Sec61β paralogs in this yeast results in heat sensitivity instead of cold sensitivity. Nevertheless, Sec61β homologs from both S. pombe and S. cerevisiae complement the respective temperature sensitivity of either yeast. This functional complementation can also be accomplished by the human homolog of the translocon β subunit, indicating that a fundamental function of Sec61β is conserved from yeast to human. Remarkably, the TMD of Sec61β homologs from S. pombe, S. cerevisiae and human are sufficient to complement the gene knockout in both fission and budding yeasts. Together, these observations indicate that the TMD of Sec61β exerts a cellular function that is conserved across species.

La sécrétion des protéines est un processus essentiel à la vie. Chez les eucaryotes, les protéines sécrétées transitent dans le réticulum endoplasmique par le pore de translocation. Le translocon est composé de trois sous-unités fondamentales nommées Sec61α, β et γ chez les mammifères, ou Sec61p, Sbh1p et Sss1p chez les levures. Tandis que le rôle des sous-unités α et γ est bien connu, celui de la sous-unité β demeure énigmatique. Plusieurs phénotypes distincts sont associés à cette protéine dans différents organismes, mais le haut niveau de conservation de séquence suggère plutôt une fonction universelle conservée. Récemment, Feng et al. (2007) ont montré que le domaine transmembranaire (TMD) de Sbh1p était suffisant pour complémenter plusieurs phénotypes associés à la délétion du gène chez Saccharomyces cerevisiae, suggérant un rôle important de cette région. L’objectif de mon projet de recherche consiste à étudier la fonction biologique de la sous-unité β du translocon et de son TMD chez Schizosaccharomyces pombe. Dans cette levure, j’ai découvert que le gène sbh1+ n’était pas essentiel à la viabilité à 30oC, mais qu’il était requis pour la croissance à basse température. La délétion de sbh1+ entraîne une sensibilité aux stress de la paroi cellulaire et une diminution de la sécrétion des protéines à 23oC. La surexpression de Sbh1p diminue elle aussi la sécrétion des protéines et altère la morphologie cellulaire. Ces phénotypes sont distincts de ceux observés chez S. cerevisiae, où la délétion des deux paralogues de Sec61β entraîne une sensibilité à haute température plutôt qu’à basse température. Malgré cela, les homologues de Sec61β de S. pombe et de S. cerevisiae sont tout deux capables de complémenter la thermosensibilité respective de chaque levure. La complémentation est possible même avec l’homologue humain de Sec61β, indiquant la conservation d’une fonction de Sec61β de la levure à l’homme. Remarquablement, le TMD de Sec61β de S. pombe, de S. cerevisiae et de l’humain sont suffisants pour complémenter la délétion génomique autant chez la levure à fission que chez la levure à bourgeons. Globalement, ces observations indiquent que le TMD de Sec61β exerce une fonction cellulaire conservée à travers les espèces. / Protein secretion is an essential biological process. In eukaryotes, secreted proteins transit into the endoplasmic reticulum through the translocon pore. The core of the translocation channel is composed of three subunits called Sec61α, β and γ in mammals, or Sec61p, Sbh1p and Sss1p in yeasts. While the role of the α and γ subunit is well understood, the function of the β subunit remains ill-defined. Although numerous species-specific phenotypes have been reported for this protein, the striking sequence conservation among species argue in favour of a universal role. Recently, Feng et al. (2007) reported the surprising finding that the transmembrane domain (TMD) of Sbh1p was sufficient to complement different functions of the entire protein in Saccharomyces cerevisiae, suggesting an important role for this region. The aim of my project was to explore the biological function of the translocon β subunit and its TMD in Schizosaccharomyces pombe. In this yeast, we found that the sbh1+ gene is unessential for viability at 30oC, but is required for growth at low temperature. Knockout of sbh1+ results in sensitivity to cell-wall stress and reduced protein secretion at 23oC. Overexpression of Sbh1p also diminishes protein secretion and results in an elongated cell shape. These phenotypes contrast with those observed S. cerevisiae, as deletion of both Sec61β paralogs in this yeast results in heat sensitivity instead of cold sensitivity. Nevertheless, Sec61β homologs from both S. pombe and S. cerevisiae complement the respective temperature sensitivity of either yeast. This functional complementation can also be accomplished by the human homolog of the translocon β subunit, indicating that a fundamental function of Sec61β is conserved from yeast to human. Remarkably, the TMD of Sec61β homologs from S. pombe, S. cerevisiae and human are sufficient to complement the gene knockout in both fission and budding yeasts. Together, these observations indicate that the TMD of Sec61β exerts a cellular function that is conserved across species.

Abstract The function of skeletal muscle is to generate force and produce movement. These tasks are carried out by long multinucleated cells, the skeletal myofibres. The membrane system and the cytoskeleton of these cells are uniquely organized to respond rapidly to neuronal stimuli and to achieve efficient contraction. In the present study the organization and distribution of selected protein/lipid based microdomains that reside in the plasma membrane and sarcoplasmic reticulum of isolated rat skeletal myofibres, were investigated. Aquaporin 4 (AQP4) water channels are arranged as higher order oligomers of several sizes in the sarcolemma and in the T tubules. These oligomers, however, were absent from many specialized micro- and- macrodomains. The distribution of AQP4 coincided with that of a highly organized protein assembly, the dystrophin glycoprotein complex (DGC), in the sarcolemma. A chimaeric venus-AQP4 was equally mobile in the T tubules and sarcolemma, but the anchoring mechanisms of the protein appeared to be different. In contrast to AQP4, the proteins resident in cholesterol and sphingolipid-based microdomains, known as rafts, also occupied DGC deficient areas, which surround the T tubule openings. Indeed, flotillin-1 rafts were located in the neck portions of the T tubules. The rafts defined by the influenza haemagglutinin (HA) also resided in DGC deficient areas, but at the borders of the DGC area. Importantly, of the raft proteins, only the localization of caveolin 3 (CAV3) was dependent on the cholesterol enriched lipid environment, as evidenced by cholesterol depletion experiments and localization studies on a non-raft associated variant of HA. The organization and distribution of membrane associated rough ER (RER) proteins were also analysed. Biochemical detergent extraction analyses and immunofluorescence staining indicated that the ER proteins were assembled as microdomains within the sarcoplasmic reticulum (SR). The microdomains were distributed throughout the SR network and they were capable of protein translocation. Taken together, skeletal myofibres comprise visually distinct microdomains both in the plasma membrane and in the SR. In the plasma membrane, different types of microdomains are not homogenously distributed and function in diverse locations. This may have important physiological implications concerning, among other things, local regulation of ion concentrations and cell signalling cascades. Different constraints ranging from protein-protein interactions to the surrounding lipid environment are important for dictating the observed distribution patterns. / Tiivistelmä Luustolihaksen toimintojen perustana ovat supistumiskykyiset lihassolut, joiden kalvorakenne on järjestynyt erityisellä tavalla ohjaamaan supistusta. Tässä tutkimuksessa analysoitiin proteiini- ja lipidiperustaisten mikroalueiden järjestäytymistä ja tähän vaikuttavia tekijöitä luustolihassolun solukalvolla sekä lihassolun sisäisessä kalvojärjestelmässä, sarkoplasmisessa verkossa (SR). Ensin analysoitiin vesikanavatyyppiä 4 (AQP4), joka oligomerisoituessaan muodostaa erikokoisia mikroalueita. Havaittiin, että AQP4-mikroalueita esiintyy kaikkialla solukalvolla lukuun ottamatta eräitä erilaistuneita mikro- ja makroalueita. AQP4-oligomeerien jakauma solukalvon lateraalisessa osassa, sarkolemmalla, noudatti dystrofiini-glykoproteiinikompleksin jakaumaa. Fluoresoivan venus-AQP4-proteiinin avulla osoitettiin, että proteiinin liikkuvuus oli samanlainen solun sisään ulottuvissa poikkiputkistoissa ja sarkolemmalla, mutta liikkuvuutta rajoittavat tekijät olivat erilaisia näissä solukalvon osissa. Toiseksi analysoitiin kolesteroli- ja sfingolipidipitoisia mikroalueita, kalvolauttoja. Flotilliini-1- ja influenssaviruksen hemagglutiniini (HA) -proteiinia sisältäviä lauttoja esiintyi vain poikkiputkien suuaukkojen alueella, mutta lauttojen jakauma oli erilainen. Lauttojen lipidiympäristöllä ei ollut vaikutusta proteiinien sijaintiin. Tämä osoitettiin kolesterolin poistokokeilla sekä kokeilla, joissa käytettiin mutatoitua HA-proteiinia, joka ei hakeudu kolesteroliympäristöön. Kaveoliini-3-proteiinin sijainti poikkeaa edellä mainituista, ja kolesterolin poisto vaikutti merkittävästi sijainnin määräytymiseen. Kolmanneksi analysoitiin, miten karkean endoplasmakalvoston proteiinit ovat järjestäytyneet SR:ssä. Havaittiin, että endoplasmiset kalvoproteiinit eivät ole homogeenisesti levittäytyneet SR-kalvostoon vaan muodostavat pieniä mikroalueita. Detergenttiuuttoanalyysit osoittivat lisäksi, että näissä mikroalueissa on erilainen lipidikoostumus kuin SR:ssä yleensä. Huomattavaa oli myös, että mikroalueet olivat toiminnallisia kaikkialla SR-kalvostossa. Tulosten perusteella luustolihassolujen kalvojärjestelmä sisältää mikroalueita, joiden jakautuminen vaikuttaa hyvin organisoituneelta. Erityisesti solukalvon mikroalueet esiintyvät tietyillä spesifeillä alueilla, joissa niiden voidaan olettaa toimivan mm. erilaisissa solusignalointitapahtumissa ja paikallisessa ionipitoisuuksien säätelyssä. Eräissä tapauksissa lipidiympäristöllä on merkitystä mikroalueiden sijainnin määräytymisessä, mutta proteiinien sitoutuminen solukalvo- tai solukalvon alaisiin rakenteisiin saattaa myös olla määräävä tekijä.

Gleich einem Etikett dient die N-Glykokosylierung vom Ribosom neu synthetisierter Proteine durch die Oligosaccharyltransferase (OST) bei der kotranslationalen Translokation in das Endoplasmatische Retikulum (ER) als Startpunkt vielschichtiger Prozessierungen. Bisher fehlte der strukturelle Nachweis, dass die OST als mit dem Ribosom assoziierten Membranprotein (RAMP) Bestandteil des auf dem proteinleitenden Kanal, dem Sec61-Komplex, basierenden Translokons ist. In dieser Arbeit berichten wir von der kryoelektronenmikroskopischen 3D-Struktur eines definierten OST-Sec61-Ribosom-Komplexes aus Saccharomyces cerevisiae bei 15,4 Å Auflösung. Dazu wurden die Komponenten (OST, Sec61 und Ribosomen mit naszierender Proteinkette) affinitätschromatographisch gereinigt und das Bindungsverhalten mit 80S-Ribosomen in vitro untersucht. Die OST band mit einer KD von 12,8 nM hochaffin und spezifisch an den bekannten Sec61-Ribosomen-Komplex. Dieser in vitro rekonstituierte trimere Komplex zeigte eine neuartige eng an das Ribosom anschließende Translokonstruktur mit zwei bisher unbekannten ribosomalen Verbindungen, einer einzigen dezentralen porenförmigen Vertiefung und zusätzlichen luminalen Bereichen. Durch das Docken eines Sec61-Homologs in einer alternativen Bindeposition sowie das Docken eines Stt3p-Homologs (der katalytischen Untereinheit der OST) und mit Hilfe der mittels (Kryo-)Negativkontrastierung gewonnenen 3D-Struktur der OST konnten Hybridmodelle erstellt werden. Daraus wurde unter Einbeziehung von bekannten molekularbiologisch gewonnenen Interaktionsdaten das 3D-Modell eines aktiven Ribosomen-gebundenen OST-Sec61-Translokons entwickelt. / Like a label, N-glycosylation by the oligosaccharyltransferase (OST) of newly synthesized proteins emerging from the ribosome while being cotranslationally translocated into the endoplasmic reticulum (ER) provides a starting point for a multitude of processes. Hitherto no structural proof has been presented, that the OST as a ribosome associated membrane protein (RAMP) is a constituent of the translocon, based at its core on the protein conducting channel (Sec61-complex). In this work we report on the 3D-structure of a defined OST-Sec61-ribosome complex from Saccharomyces cerevisiae by cryo-electron microscopy at 15.4 Å resolution. Thereto, the components (OST, Sec61, ribosome nascent chain complexes) have been purified by affinity chromatography and the binding of 80S-ribosomes has been checked in vitro. The OST bound with high affinity by a KD of 12.8 nM specifically to the established Sec61-ribosome complex. This trimeric complex reconstituted in vitro exhibits a new kind of tightly bound ribosomal translocon showing two hitherto unknown connections to the ribosome, a single off-center pore-like indentation and an additional luminal domain. By docking of a Sec61 homologue at an alternative binding position plus the docking of a Stt3p homologue (the catalytic subunit of the OST) and by means of the 3D-structure of the OST using the (cryo-)negative staining technique, hybrid models could be created. Consequently, integrating known interaction data from molecular biology experiments has been used to develop a 3D-model of an active ribosome-bound OST-Sec61-translocon.