Browsing by Subject "Biomembran"

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Intrazelluläres Trafficking des intestinalen Anionenaustauschers Down-Regulated in Adenoma (DRA;SLC26A3)
(2011) Lissner, Simone; Graeve, Lutz
Electroneutral NaCl absorption occurs from the small intestine to the distal colon. This ion exchange is preferentially mediated by DRA and NHE3. Knockout mice, which suffer from chronic diarrhea, as well as the human genetic disorder congenital chloride diarrhea, in which a nonfunctional DRA leads to life-threatening diarrhea emphasize the importance of these two transporters. To elucidate this defective NaCl absorption it is necessary to understand the physiological regulation of these two transport proteins within enterocytes as well as the responsible extra- and intracellular signal transduction pathways. Both transport proteins interact with PDZ adaptor proteins of the NHERF family. Furthermore, both exchangers are partially localized within lipid rafts. The situation for NHE3 is complex in that its lipid raft localization is not only necessary for its normal activity but also for its basal and stimulated trafficking. Lipid rafts are involved in PI3-kinase dependent exocytosis of NHE3. Since the function of NHE3 and DRA appears to be regulated in parallel the function of DRA maybe depends on its rafts association as well. Thus the first objective of this thesis was to investigate whether the lipid raft association of DRA is essential for the surface expression and transport activity of DRA and also to analyze whether DRA is inserted into the plasma membrane in a PI3-kinase and lipid raft dependent manner. The present data show that: (A) Disruption of lipid raft integrity leads to functional inhibition and decreased cell surface expression of DRA. In HEK cells the inhibition of DRA activity as well as the decreased cell surface expression are entirely dependent on the presence of the PDZ interaction motif of DRA. In Caco-2/BBE cells on the other hand only part of the inhibition of DRA activity by disruption of raft integrity depends on the ability of DRA to interact with PDZ adaptor proteins. (B) Basal activity as well as basal surface expression of DRA depend on PI3-kinase activity in a way that requires the ability of DRA to interact with PDZ adaptor proteins. (C) Lipid rafts and PI3-kinase are situated along the same pathway, where DRA is present in lipid rafts before it is inserted into the plasma membrane. However, the inhibition of PI3-kinase has no influence on the raft association of DRA. Furthermore, the disruption of raft integrity does not inhibit the PI3-kinase activity. Based on these findings a model can be established as follows: DRA is present in lipid rafts in an intracellular fraction. Insertion into the plasma membrane from this intracellular compartment requires the interaction with one (or several) PDZ adaptor proteins, raft integrity and the action of PI3-kinase. To characterize the interplay between PI3-kinase, raft association and PDZ interaction of DRA with its insertion into the plasma membrane the recycling pathway of DRA was then investigated. The generated data show that the proteolytic degradation of DRA-ETKFminus occurs faster than the degradation of wild type DRA. Endosomal distribution of DRA depends on its PDZ-binding motif. The sorting process from early to recycling endosomes depends on the interaction of DRA with one or several PDZ adaptor proteins. Expression of dominant negative Rab11a leads to a decreased surface expression and transport activity of DRA. In conclusion, it was shown in this thesis that an intense interplay between PDZ interaction, lipid raft association, PI3-kinase and the activity and surface expression of DRA exists. It was also shown that the endosomal distribution of DRA depends on its PDZ-binding motif. Finally, it was demonstrated that DRA is recycled to the plasma membrane by Rab11a-enriched recycling endosomes.
Posttranslationale Modifikationen der IL-6-Typ-Zytokin-Rezeptoren gp130 und LIFR und ihr Einfluss auf die Assoziation mit Detergenz-resistenten Membranmikrodomänen (DRM)
(2008) Ziegler, Inna; Graeve, Lutz
Post-translational modification of proteins is an important event in the regulation of cellular functions. Glycosylation or palmitoylation, but also ligand binding can affect the localization of proteins in membrane microdomains and thus affect signal transduction. The aim of this study was to analyze how posttranslational modifications of LIFR and the common signal transducer gp130 impact the translocation to detergent resistant membranes (DRMs, lipid rafts). Palmitoylation of cysteine residues within the transmembrane domain of a protein is considered to be one process that assists in the localization of proteins to DRMs. Gp130 has two cysteine residues C711 and C725 in its transmembrane domain. My studies indicate that these cysteine residues have no significant influence on lipid raft association of gp130. Contrary to our expectations, after isolation of DRMs with Brij 58 and Triton X-100 an increase of raft association of the C->A-mutants was detected. Partial DRM association of LIFR was confirmed by using Brij 58 and Triton X-100 protocols. Furthermore, two different N-glycosylation types of that receptor could be detected. The mannose-rich (precursor) species is preferentially found in non-DRMs and is degraded by Endo-Glycosidase Hf. The hybrid-type (mature) tends towards an association with DRMs. My results indicate that only the mature-type of LIFR was phosphorylated after LIF binding to the receptor complex in 3T3-L1 and HepG2 cells. Combined with other data from our workgroup these findings suggest that only the mature-type of LIFR is expressed at the plasma membrane surface and involved in signal transduction. After stimulation with LIF an increase of LIFR tyrosine phosphorylation was observed in DRMs in HepG2 cells. However, phosphorylation of gp130 was detected only in non-DRMs fractions after stimulation with LIF. The inconsistency of these results can be explained with methodical problems. Furthermore, the translocation of phosphorylated receptors described above could not confirmed in 3T3-L1 cells. In this cell line, the activation of gp130 and LIFR occurs in detergent-resistant membranes. These findings indicate differences between cell lines with respect to receptor activation and translocation within the plasma membrane on the one hand and demonstrate a differential sensitivity of raft subdomains to extraction by different detergents on the other hand.
Untersuchungen zur autonomen und YidC-vermittelten Membraninsertion von Pf3 coat-Protein mit Hilfe Fluoreszenz-spektroskopischer Einzelmolekülmessungen
(2011) Schönbauer, Anne-Kathrin; Kuhn, Andreas
Pf3 coat is the capsid protein of the bacteriophage Pf3. The phage leaves the host cell by continuous extrusion without damaging the cell. The protein itself consists of 44 amino acid residues and has a rod-like shape. Because of its simple structure, the protein needs only the help of the insertase YidC to insert into the bacterial inner membrane. 3L-Pf3 coat, a protein mutant with three additional leucine residues in the center of the transmembrane region (TMD), has an increased hydrophobicity. It is independent of YidC and inserts into the membrane autonomously (Serek et al., 2004). In this work, a newly developed physical method was used to find out whether the elongation or the increased hydrophobicity accounts for the autonomous insertion of the protein. For this reason, two new protein mutants were constructed. Each mutant has only one of the changed properties of the 3L-Pf3 coat protein: GAT-Pf3 coat has an elongated TMD with three additional residues (glycine, alanin and threonine). The second mutant, 2M-Pf3 coat, shows an increased hydrophobicity due to the substitution of two alanine residues by two methionine residues at the positions 30 and 31. So it had an increased hydrophobicity like 3L-Pf3 coat. The above mentioned proteins, wt-Pf3 coat and its mutants, were modified with a fluorescent label to follow the proteins with optical methods. The Proteins were first modified with a single cysteine and then labeled by a fluorescent marker, Atto520 maleimid. Proteins with a labeled N-terminal tail were called NC-Pf3 coat, whereas CC-Pf3 coat had a labeled C-terminal tail. In addition, the orientation of the protein in the membrane was identified by quenching the fluorescence of the NC- and CC- labeled proteins. A new method employing single molecules was developed using fluorescence correlation spectroscopy. This method allows real time observations of binding and insertion of the protein into semisynthetical liposomes. By using fluorescent quenching the membrane insertion and binding were distinguished. It became clear that both the elongation of the TMD as well as an increased hydrophobicity play a crucial role in the autonomous insertion of the protein into the membrane. Therefore, the interaction between the hydrophobic region of the protein and the hydrophobic core region of the membrane is important for the binding of the protein and its insertion into the membrane.