Dissertation (Ph.D.).--University of Texas Graduate School of Biomedical Sciences at San Antonio, 2007. / Vita. Includes bibliographical references.

Diss. (sammanfattning) Umeå : Univ., 2004. / Härtill 3 uppsatser.

No description available.

Vesicular traffic along the biosynthetic and endocytic pathways is essential for homeostasis of eukaryotic cells. However, it raised the question of how the proteins characteristic for each compartment are transported to their destination (Bonifacino and Glick, 2004). This study is especially focusing on the connection between the Golgi apparatus and the endosomal compartment, mediated by two parallel trafficking pathways regulated by the clathrin adaptors AP-1A and AP-3 (Owen et al., 2004). Typical cargo molecules sorted along the AP-1A regulated pathway are mannose 6-phosphate receptors (MPRs) (Ghosh et al., 2003) or the gpI envelop glycoprotein of the Vesicular Zoster virus (Alconada et al., 1996), while sorting of lysosomal membrane proteins like Lamp-1 and LimpII is AP-3 regulated (Eskelinen et al., 2003). To study how AP-1A and AP-3 coats are stabilized on membranes and to identify the protein networks involved, a liposome based in vitro assay that recapitulates the fidelity of protein sorting in vivo was developed and combined with proteomic screens. Therefore, liposomes carrying cytoplasmic domains of gpI or Lamp-1/LimpII were used as affinity matrix to recruit selectively AP-1A or AP-3 and associated protein machineries. The coated liposomes were then analyzed by mass spectrometry. Using the in vitro recruitment assay, it was possible to demonstrate that efficient and selective recruitment of AP-1A and AP-3 coats depends on the presence of several low affinity binding sites on membranes. Thus, AP-1A and AP-3 recognize their target membranes by activated Arf1 GTPases, organelle specific phosphoinositides, PI-4P and PI-3P respectively, and distinct cargo molecules carrying intact signals in their cytoplasmic domains. The implication of PI-3P in AP-3 recruitment was further supported by in vivo experiments. During the biochemical characterization of the assay, several lines of evidence indicated that cargo tails containing intact sorting signals stabilize not only AP-1A and AP-3 coats on membranes but also influence the membrane recruitment of Arf1. It is possible that cargo molecules indirectly drive an Arf1 amplification loop, thereby ensuring efficient AP coat assembly. The proteomic screens identified protein networks of ≈40 proteins selectively recruited on AP-1A coated structures. The most appealing result of the analysis was the presence of two additional protein machineries, one involved in actin nucleation the other involved membrane fusion. More precisely, the AP-1A analysis identified the selective recruitment of the AP-1A subunits and interacting molecules (clathrin, g-synergin), Arf1 and Arf1 effectors (Big2, Git1), Rac1 including Rac1 effectors (b-PIX, RhoGEF7) and a Rac1 dependent actin nucleation machinery (Wave/Scar complex, Arp2/3 complex, associated effectors) as well as members of a Rab machinery (Rab11, Rab14). This finding was further supported by in vivo colocalization studies of the AP-1A cargo CI-MPR with CYFIP2, a protein of the Wave/Scar complex, and the localization of Big2 and Git1 on Rab11 positive membranes (Matafora et al., 2001; Shin et al., 2004). The biochemical characterization revealed that the stabilization of AP-1A coats, most probably driven by cargo molecules that stabilize AP-1A and Arf1 on membranes, leads as well to the stabilization of the two other machineries. Thus, the results support the notion that cargo sorting, vesicular movement and membrane fusion are coordinated during early steps of vesicular traffic. In analogy, the proteomic screens on AP-3 coated structures identified as well ≈40 selectively recruited proteins, which constituted a similar supramolecular network of protein machineries involved in coat formation, action nucleation and membrane fusion via Rab proteins. Thus, beside the AP-3 coat including the AP-3 subunits, Arf1 and Arf effectors (Big1, ARAP1, AGAP1), members of the septin family involved in actin rearrangements and most of the already described effectors of Rab5 microdomains (EEA1, Rabaptin-5, Rabex-5, Vps45) involved in early endosomal dynamics were selectively recruited together with Rab5 and Rab7. Thus, the proteomic analysis of AP-1A and AP-3 coated structures suggest that both AP coats use similar principles - coats, actin nucleation devices and Rab fusion machineries - to assemble supramolecular structures needed for membrane traffic. Although we do not have the ultimate proves yet, it seems as AP-1A and AP-3 use different members of subcomplexes, hence different GTPase effectors, different actin nucleation machineries and different Rab GTPases, to regulate their specific transport pathways and to link the different protein machineries. The proteomic analysis revealed for example that they probably use different Arf and Rho GTPase effectors to link the coat with actin nucleation. However, this has to be proven experimentally. In order to understand the networks of protein interactions, bioinformatic tools were used as a first approach. Even though some clues about the overall organization of the supramolecular protein complexes were provided, the direct links to the Rab machinery are still elusive. Maybe the proteins with thus far unknown functions could be involved. The biochemical analysis, especially the role of PIPs, and the Rab GTPases identified in the context of AP-1A and AP-3, provide indications about AP-1A and AP-3 function in vivo. The results could be interpreted in a way that AP-1A functions either in traffic from PI-4P positive membranes towards Rab11/Rab14 positive membranes or AP-1A coats assemble on PI-4P and Rab11 or Rab14 positive membranes, hence, TGN to endosomes traffic. The same holds true for AP-3, the results either suggest AP-3 mediates traffic from PI-3P positive towards Rab5/Rab7 positive membranes or they could be interpreted in a way that AP-3 assembles on PI-3P and Rab5 positive membranes for subsequent transport to Rab7 positive membranes, thus traffic from early to late endosomes. Overall, the results of this thesis research provided important insight into the formation of AP-1A and AP-3 coated structures and the potential interconnection between AP coats, actin nucleation and membrane fusion machineries. Alconada, A., U. Bauer, and B. Hoflack. 1996. A tyrosine-based motif and a casein kinase II phosphorylation site regulate the intracellular trafficking of the varicella-zoster virus glycoprotein I, a protein localized in the trans-Golgi network. Embo J. 15:6096-110. Bonifacino, J.S., and B.S. Glick. 2004. The mechanisms of vesicle budding and fusion. Cell. 116:153-66. Eskelinen, E.L., Y. Tanaka, and P. Saftig. 2003. At the acidic edge: emerging functions for lysosomal membrane proteins. Trends Cell Biol. 13:137-45. Ghosh, P., N.M. Dahms, and S. Kornfeld. 2003. Mannose 6-phosphate receptors: new twists in the tale. Nat Rev Mol Cell Biol. 4:202-12. Matafora, V., S. Paris, S. Dariozzi, and I. de Curtis. 2001. Molecular mechanisms regulating the subcellular localization of p95-APP1 between the endosomal recycling compartment and sites of actin organization at the cell surface. J Cell Sci. 114:4509-20. Owen, D.J., B.M. Collins, and P.R. Evans. 2004. Adaptors for clathrin coats: structure and function. Annu Rev Cell Dev Biol. 20:153-91. Shin, H.W., N. Morinaga, M. Noda, and K. Nakayama. 2004. BIG2, a guanine nucleotide exchange factor for ADP-ribosylation factors: its localization to recycling endosomes and implication in the endosome integrity. Mol Biol Cell. 15:5283-94.

Vesicular traffic along the biosynthetic and endocytic pathways is essential for homeostasis of eukaryotic cells. However, it raised the question of how the proteins characteristic for each compartment are transported to their destination (Bonifacino and Glick, 2004). This study is especially focusing on the connection between the Golgi apparatus and the endosomal compartment, mediated by two parallel trafficking pathways regulated by the clathrin adaptors AP-1A and AP-3 (Owen et al., 2004). Typical cargo molecules sorted along the AP-1A regulated pathway are mannose 6-phosphate receptors (MPRs) (Ghosh et al., 2003) or the gpI envelop glycoprotein of the Vesicular Zoster virus (Alconada et al., 1996), while sorting of lysosomal membrane proteins like Lamp-1 and LimpII is AP-3 regulated (Eskelinen et al., 2003). To study how AP-1A and AP-3 coats are stabilized on membranes and to identify the protein networks involved, a liposome based in vitro assay that recapitulates the fidelity of protein sorting in vivo was developed and combined with proteomic screens. Therefore, liposomes carrying cytoplasmic domains of gpI or Lamp-1/LimpII were used as affinity matrix to recruit selectively AP-1A or AP-3 and associated protein machineries. The coated liposomes were then analyzed by mass spectrometry. Using the in vitro recruitment assay, it was possible to demonstrate that efficient and selective recruitment of AP-1A and AP-3 coats depends on the presence of several low affinity binding sites on membranes. Thus, AP-1A and AP-3 recognize their target membranes by activated Arf1 GTPases, organelle specific phosphoinositides, PI-4P and PI-3P respectively, and distinct cargo molecules carrying intact signals in their cytoplasmic domains. The implication of PI-3P in AP-3 recruitment was further supported by in vivo experiments. During the biochemical characterization of the assay, several lines of evidence indicated that cargo tails containing intact sorting signals stabilize not only AP-1A and AP-3 coats on membranes but also influence the membrane recruitment of Arf1. It is possible that cargo molecules indirectly drive an Arf1 amplification loop, thereby ensuring efficient AP coat assembly. The proteomic screens identified protein networks of ≈40 proteins selectively recruited on AP-1A coated structures. The most appealing result of the analysis was the presence of two additional protein machineries, one involved in actin nucleation the other involved membrane fusion. More precisely, the AP-1A analysis identified the selective recruitment of the AP-1A subunits and interacting molecules (clathrin, g-synergin), Arf1 and Arf1 effectors (Big2, Git1), Rac1 including Rac1 effectors (b-PIX, RhoGEF7) and a Rac1 dependent actin nucleation machinery (Wave/Scar complex, Arp2/3 complex, associated effectors) as well as members of a Rab machinery (Rab11, Rab14). This finding was further supported by in vivo colocalization studies of the AP-1A cargo CI-MPR with CYFIP2, a protein of the Wave/Scar complex, and the localization of Big2 and Git1 on Rab11 positive membranes (Matafora et al., 2001; Shin et al., 2004). The biochemical characterization revealed that the stabilization of AP-1A coats, most probably driven by cargo molecules that stabilize AP-1A and Arf1 on membranes, leads as well to the stabilization of the two other machineries. Thus, the results support the notion that cargo sorting, vesicular movement and membrane fusion are coordinated during early steps of vesicular traffic. In analogy, the proteomic screens on AP-3 coated structures identified as well ≈40 selectively recruited proteins, which constituted a similar supramolecular network of protein machineries involved in coat formation, action nucleation and membrane fusion via Rab proteins. Thus, beside the AP-3 coat including the AP-3 subunits, Arf1 and Arf effectors (Big1, ARAP1, AGAP1), members of the septin family involved in actin rearrangements and most of the already described effectors of Rab5 microdomains (EEA1, Rabaptin-5, Rabex-5, Vps45) involved in early endosomal dynamics were selectively recruited together with Rab5 and Rab7. Thus, the proteomic analysis of AP-1A and AP-3 coated structures suggest that both AP coats use similar principles - coats, actin nucleation devices and Rab fusion machineries - to assemble supramolecular structures needed for membrane traffic. Although we do not have the ultimate proves yet, it seems as AP-1A and AP-3 use different members of subcomplexes, hence different GTPase effectors, different actin nucleation machineries and different Rab GTPases, to regulate their specific transport pathways and to link the different protein machineries. The proteomic analysis revealed for example that they probably use different Arf and Rho GTPase effectors to link the coat with actin nucleation. However, this has to be proven experimentally. In order to understand the networks of protein interactions, bioinformatic tools were used as a first approach. Even though some clues about the overall organization of the supramolecular protein complexes were provided, the direct links to the Rab machinery are still elusive. Maybe the proteins with thus far unknown functions could be involved. The biochemical analysis, especially the role of PIPs, and the Rab GTPases identified in the context of AP-1A and AP-3, provide indications about AP-1A and AP-3 function in vivo. The results could be interpreted in a way that AP-1A functions either in traffic from PI-4P positive membranes towards Rab11/Rab14 positive membranes or AP-1A coats assemble on PI-4P and Rab11 or Rab14 positive membranes, hence, TGN to endosomes traffic. The same holds true for AP-3, the results either suggest AP-3 mediates traffic from PI-3P positive towards Rab5/Rab7 positive membranes or they could be interpreted in a way that AP-3 assembles on PI-3P and Rab5 positive membranes for subsequent transport to Rab7 positive membranes, thus traffic from early to late endosomes. Overall, the results of this thesis research provided important insight into the formation of AP-1A and AP-3 coated structures and the potential interconnection between AP coats, actin nucleation and membrane fusion machineries. Alconada, A., U. Bauer, and B. Hoflack. 1996. A tyrosine-based motif and a casein kinase II phosphorylation site regulate the intracellular trafficking of the varicella-zoster virus glycoprotein I, a protein localized in the trans-Golgi network. Embo J. 15:6096-110. Bonifacino, J.S., and B.S. Glick. 2004. The mechanisms of vesicle budding and fusion. Cell. 116:153-66. Eskelinen, E.L., Y. Tanaka, and P. Saftig. 2003. At the acidic edge: emerging functions for lysosomal membrane proteins. Trends Cell Biol. 13:137-45. Ghosh, P., N.M. Dahms, and S. Kornfeld. 2003. Mannose 6-phosphate receptors: new twists in the tale. Nat Rev Mol Cell Biol. 4:202-12. Matafora, V., S. Paris, S. Dariozzi, and I. de Curtis. 2001. Molecular mechanisms regulating the subcellular localization of p95-APP1 between the endosomal recycling compartment and sites of actin organization at the cell surface. J Cell Sci. 114:4509-20. Owen, D.J., B.M. Collins, and P.R. Evans. 2004. Adaptors for clathrin coats: structure and function. Annu Rev Cell Dev Biol. 20:153-91. Shin, H.W., N. Morinaga, M. Noda, and K. Nakayama. 2004. BIG2, a guanine nucleotide exchange factor for ADP-ribosylation factors: its localization to recycling endosomes and implication in the endosome integrity. Mol Biol Cell. 15:5283-94.

No description available.

MicroRNAs (miRNAs) sind kurze nicht-kodierende RNAs, die auf posttranskriptionaler Ebene die Genexpression hemmen. Dafür bilden miRNAs Ribonukleoprotein-Komplexe, deren Kernbestandteile aller Bilateria Argonaute (AGO) und GW182 /TNRC6 Proteine sind. GW182 / TNRC6-Proteine rekrutieren CCR4-NOT-Deadenylasen über kurze Tryptophan-reiche Motive (W-Motive), welche additiv wirken und fördern so die translationale Repression und den Abbau von Ziel-mRNAs. Um mehr über die Mechanismen der miRNA-abhängigen Genrepression zu erfahren, habe ich W-Motiv-abhängige Interaktionspartner humaner TNRC6C Proteine bestimmt. Hierzu habe ich, mithilfe von quantitativer Massenspektrometrie, das Interaktom von wildtyp TNRC6C Proteinen mit dem von TNRC6C Proteinen, deren W-Motive mutiert wurden, verglichen. Neben bekannten Interaktionspartnern, wie Untereinheiten des CCR4-NOT Komplexes, habe ich Komponenten von Clathrin-Vesikeln (CCVs), Stoffwechsel assoziierte Enzyme, mitochondriale Proteine, RNA Helikasen, Kinasen und Phosphatasen mit potentiellen Funktionen in der miRNA-assoziierten Repression identifiziert. Die im ersten Teil dieser Studie vorgestellten Ergebnisse legen nahe, dass CCVs die Speicherung oder das Recycling von TNRC6 und AGO Proteinen vermitteln können und somit das miRNA-Silencing modulieren. Der zweite Teil dieser Studie befasst sich mit der Konservierung von miRNA vermitteltem Gen-Silencing in Cnidaria (Nematostella vectensis), welche sich vor 600 Millionen Jahren von der Ahnenreihe der Metazoa abspalteten. Hier zeige ich anhand humaner Zellen, dass Nematostella GW182, ähnlich wie in Bilateria, von AGO rekrutiert wird und nachfolgend in der Repression der mRNA fungiert, was darauf hinweist, dass dieser Mechanismus der miRNA-vermittelten Geninhibition bereits in den letzten gemeinsamen Vorfahren von Cnidaria und Bilateria aktiv war. / MicroRNAs (miRNAs) are short non-coding RNAs that act as post-transcriptional repressors of gene expression. To function miRNAs are assembled in ribonucleoprotein complexes, whose core components in bilaterian animals are Argonaute (AGO) and GW182/TNRC6 proteins. GW182/TNRC6 proteins additively recruit CCR4-NOT deadenylases via short tryptophan-containing motifs (W-motifs), thereby promoting translational repression and the decay of target mRNAs. To gain deeper insights into the mechanisms of miRNA silencing I determined the W-motif-specific interactome of human TNRC6C proteins. Using Stable Isotope Labeling by Amino acids in Cell Culture (SILAC) coupled to affinity purification and Mass Spectrometry (MS) I identified proteins enriched with wild type TNRC6C as compared to two mutants with disrupted W-motifs. Besides known functional interactors, such as subunits of the CCR4-NOT complex, I identified several components of clathrin-coated vesicles (CCVs), metabolic enzymes, mitochondrial proteins, RNA helicases, kinases, and phosphatases with potential functional roles in miRNA-mediated repression. The results presented in the first part of this thesis indicate that CCVs may mediate the storage or recycling of TNRC6 and AGO proteins, thus modulating miRNA silencing. The second part of the thesis addressed the conservation of the mechanisms of miRNA silencing via W-motifs in the cnidarian Nematostella vectensis, separated by 600 million years from other Metazoa. Using cultured human cells, I showed that similarly to bilaterians, GW182 in Nematostella is recruited to the miRNA repression complex via interaction with AGO proteins, and functions downstream to repress mRNA, indicating that this mechanism of miRNA-mediated silencing was already active in the last common ancestor of Cnidaria and Bilateria.