Charakterisierung der großen GTPase Myxovirus Resistance Protein 1 (Mx1) im intrazellulären Proteintransport

Der intrazelluläre Proteintransport in polaren Epithelzellen basiert zunächst auf der Seite der zu transportierenden Moleküle aus einem komplexen Code von Sortiersignalen, Proteinmodifikationen und einer Assoziation mit spezifischen Lipidkompositionen. Dem gegenüber steht auf der anderen Seite eine...

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Bibliographic Details
Main Author: Hoff, Florian
Contributors: Jacob, Ralf (Prof. Dr.) (Thesis advisor)
Format: Doctoral Thesis
Published: Philipps-Universität Marburg 2015
Online Access:PDF Full Text
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The intracellular protein transport in polarized epithelial cells is based, in view of the transported cargo molecules, on a complex code of sorting signals, protein modifications and an association with specific lipid compositions. On the other side exists a highly specialized cellular transport machinery, consisting of a diverse set of various proteins and cytoskeletal components, which is able to decrypt this code and transports the cargo molecules to their particular destination. In search of yet unknown factors in protein transport, the large GTPase Myxovirus Resistance Protein 1 (Mx1) was discovered on vesicles of the biosynthetic lipid raft independent pathway in polarized epithelial cells. Mx1 is generally known as an interferon induced protein, which is synthesized after viral infections and which provides an antiviral immune response. The precise mechanism behind this antiviral activity is so far unknown. Different publications reveal that Mx1 is able to mislocalize viral components within the cell, which makes them unavailable for viral replication. Thus a possible function of Mx1 in the intracellular transport system is conceivable and the research focus of this dissertation. Mx1 is constitutively expressed in polarized canine kidney epithelial cells (MDCK cells) without interferon induction. In this cell line, the large GTPase was associated with post-Golgi vesicular membranes, which were positive for the neurotrophin receptor p75NTR and lipid raft independently transported towards the apical domain. The reduction of Mx1 synthesis via RNA interference demonstrated that Mx1 was not simply associated with these vesicles, but it also performed a role in their transport. Mx1 depletion led to significantly less p75NTR at the apical plasma membrane. Furthermore the secretion of the two soluble peptides galectin-3 and gp80 was also significantly reduced. The Mx1 knockdown did not result in an altered appearance of these three proteins at the basolateral membrane, but rather in a decreased efficiency of apical transport. With the help of immunofluorescence and the Proximity Ligation Assay (PLA), Mx1 was localized to membranes of endosomal origin. There, the large GTPase colocalized with galectin-3, a lectin which is essential for the correct sorting of lipid raft independent proteins. In addition Mx1 interacted directly with the tubulin- and actin cytoskeleton and the PLA showed a close proximity to tubulin dependent motor proteins. The precise function of Mx1 in intracellular transport is not yet understood. Because of its affiliation to the dynamin superfamily and structural homologies to classical dynamins, a function in fission of transport vesicles at the post- Golgi endosomal system is possible. The analysis of Mx1 expression revealed that it is not only constitutively synthesized in MDCK cells, but also present in other cell lines without interferon induction. This work demonstrates for the first time a role of Mx1 in biosynthetic protein transport. The presented observations indicate that Mx1 is not only an antiviral factor, but rather generally involved in intracellular transport mechanisms.