Thesis (PhD) - Swinburne University of Technology, Faculty of Life and Social Sciences, Brain Sciences Institute, 2008. / A thesis for Doctorate of Philosophy, Brain Sciences Institute, Faculty of Life and Social Sciences, Swinburne University of Technology - 2008. Typescript. Includes bibliographical references (p. 193-250).

Vesicle trafficking provides the solution to the 'sorting problem' - how the eukaryotic cell maintains the distinct identities, and thus functional properties, of its membrane-bound organelles. During vesicle trafficking, proteins are selectively sorted into membrane bound transport intermediates by vesicle adaptors, which include those of the highly conserved adaptor protein (AP) complex family. Each AP complex has a distinct subcellular localisation and functions in the sorting of a specific subset of transmembrane cargo proteins. Adaptor protein complex 4 (AP-4) is one of the more recently identified AP complexes, whose function has largely remained elusive. In humans, AP-4 deficiency causes a severe neurological disorder, suggesting an important role in neuronal development and homeostasis. However, the pathomechanisms that underly the neuronal pathology in AP-4 deficiency are currently unknown. AP-4 is proposed to function in protein sorting at the trans-Golgi network (TGN), so AP-4 deficiency can be thought of as a disease of missorting. The aim of this study was to apply unbiased global proteomic approaches to define the composition of AP-4 vesicles and to identify physiological cargo proteins of the AP-4 pathway. Using 'Dynamic Organellar Maps' and comparative analysis of vesicle-enriched fractions from wild-type and AP-4-depleted cells, three ubiquitously expressed transmembrane cargo proteins, ATG9A, SERINC1 and SERINC3, were found to be mislocalised in AP-4-deficient cells. Two novel cytosolic AP-4 accessory proteins, RUSC1 and RUSC2, were also identified. Further proteomic analyses confirmed the interactions between these proteins. AP-4 deficiency was found to cause missorting of ATG9A in diverse cell types, including patient derived cells, as well as dysregulation of autophagy. RUSC2 facilitates the transport of AP-4-derived, ATG9A and SERINC-positive vesicles from the TGN to the cell periphery. These vesicles cluster in close association with autophagosomes, suggesting they are the 'ATG9 reservoir' required for autophagosome biogenesis. This study uncovers ATG9A trafficking as a ubiquitous function of the AP-4 pathway. Furthermore, it provides a potential molecular pathomechanism of AP-4 deficiency, through dysregulated spatial control of autophagy.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21617号 / 医博第4423号 / 新制||医||1033(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 羽賀 博典, 教授 川口 義弥, 教授 妹尾 浩 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

EBAG9 (estrogen receptor-binding fragment-associated gene 9) hat als unabhängiger prognostischer Marker viel Aufmerksamkeit erregt, da in einigen Tumoren hohe Expressionsraten und Tumorentwicklung korrelieren. In diesen Fällen ist eine hohe EBAG9 Expression häufig mit einer schlechten klinischen Prognose verbunden. EBAG9 ist ein ubiquitär exprimiertes Golgi Protein. Aktuelle Daten demonstrieren, dass es in sekretorischen Zellen an der regulierten Exozytose und an der zytotoxischen Funktion von Lymphozyten beteiligt ist. In epithelialen Zellen führt es zur Generierung von Tumor-assoziierten O-Glykanen, welche ein Erkennungsmerkmal vieler Krebsarten sind. In dieser Arbeit wurde der pathogenetische Zusammenhang zwischen EBAG9 Expression und der Veränderung des zellulären Glykoms untersucht. Um einen tieferen Einblick in die zelluläre Funktion von EBAG9 in epithelialen Zellen zu gewinnen, wurden Zellen mit tumorähnlicher EBAG9 Expression verwendet. Innerhalb dieser Arbeit wurde demonstriert, dass EBAG9 mit anterograden COPI Vesikeln assoziiert und zwischen dem ER-Golgi intermediären Kompartiment und cis-Golgi pendelt. EBAG9 verursacht eine Verzögerung des anterograden Transportes vom ER zum Golgi und verändert die Lokalisation von Komponenten der ER Qualitätskontrolle und des Glycosylierungsapparates. Auf der anderen Seite beschleunigt die verminderte Expression von EBAG9 den Proteintransport durch den Golgi und verstärkt die Aktivität von Mannosidase II. Mechanistisch betrachtet verhindert EBAG9 die Rekrutierung von ArfGAP1 an die Membran. Dies beeinträchtigt das Auflösen der COPI Vesikelhülle und somit die Fusion von Vesikeln am cis-Golgi. Damit agiert EBAG9 in epithelialen Zellen als negativer Regulator des COPI-abhängigen ERGolgi Transportes und stellt damit ein neues phatogenetisches Prinzip dar, bei dem die Beeinflussung des intrazellulären Transportes zu der Entstehung von Tumor-assoziierten Glykanen führt. / The estrogen receptor-binding fragment-associated gene 9 (EBAG9) has received increased attention as an independent prognostic marker for disease-specific survival since in some human tumor entities high expression levels correlate with tumor progression and poor clinical prognosis. Interestingly, EBAG9 was identified as an ubiquitously expressed Golgi protein. Recent data demonstrate an involvement in regulated exocytosis in secretory cells and the cytotoxic functions of lymphocytes. However, EBAG9 is expressed in essentially all mammalian tissues, and in epithelial cells it has been identified as a modulator of tumorassociated O-linked glycan expression, a hallmark of many carcinomas. This thesis addresses the pathogenetic link between EBAG9 expression and the alteration of the cellular glycome. To gain further insights into the cellular functions of EBAG9 in epithelial cells, tumor-associated EBAG9 overexpression was mimicked in living cells. It was demonstrated that EBAG9 associates with anterograde COPI-coated carriers and shuttles between the ER-Golgi intermediate compartment and cis-Golgi stacks. EBAG9 overexpression imposes a delay in anterograde ER-to-Golgi transport and mislocalizes components of the ER quality-control and glycosylation machinery. Conversely, EBAG9 downregulation accelerates glycoprotein transport through the Golgi and enhances mannosidase activity. Functionally, EBAG9 impairs ArfGAP1 recruitment to membranes and consequently, interferes with the disassembly of the coat lattice at the cis-Golgi prior to fusion. Thus, EBAG9 acts as a negative regulator of a COPI-dependent ER-to-Golgi transport pathway in epithelial cells and represents a novel pathogenetic principle in which interference with intracellular membrane trafficking results in the emergence of a tumor-associated glycome.