Biogenesis of peroxisomes in mammalian cells: Characterization of the Pex11 proteins and their role in peroxisomal growth and division

Peroxisomes are multifunctional organelles involved in various metabolic processes. Peroxisomal malfunctions lead to numerous mostly severe disorders, rendering perox-isomes essential for human health and development. Peroxisomal abundance can be adjusted to the cellular needs, since peroxisomes hav...

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Gespeichert in:
1. Verfasser: Delille, Hannah Katharina
Beteiligte: Jacob, Ralf (Prof. Dr. ) (BetreuerIn (Doktorarbeit))
Format: Dissertation
Veröffentlicht: Philipps-Universität Marburg 2011
Klinische Zytobiologie und Zytopathologie
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Zusammenfassung:Peroxisomes are multifunctional organelles involved in various metabolic processes. Peroxisomal malfunctions lead to numerous mostly severe disorders, rendering perox-isomes essential for human health and development. Peroxisomal abundance can be adjusted to the cellular needs, since peroxisomes have the capacity to proliferate or to be degraded. Peroxisomes multiply by growth and division, but can also form de novo via the endoplasmic reticulum. Peroxisomal growth and division is a multistep process involving peroxisome elongation, constriction and final fission. The molecular components and mechanisms mediating the formation, growth, division and dynamics of peroxisomes are far from being understood. Pex11pβ, DPL1 and hFis1 were previously identified as the first molecular components involved in proliferation and division of peroxisomes in mammals. Pex11pβ mediates peroxisome elongation, while hFis1 serves as membrane adaptor for DLP1 responsible for division of the organelles. Surprisingly, DLP1 and hFis1 are involved in both mitochondrial and peroxisomal division. Aim of this study was to further reveal the molecular mechanisms of peroxisomal proliferation and division. First, the dual targeting of hFis1, a tail-anchored protein, was studied. It was demon-strated for the first time that peroxisomal but not mitochondrial targeting of hFis1 de-pends on Pex19p, a peroxisomal import factor. An essential binding region for Pex19p was located within the last 26 C-terminal amino acids of hFis1. The basic amino acids in the very C-terminus are not essential for Pex19p binding and peroxisomal targeting but are instead required for mitochondrial targeting. Since overexpression of Pex19p alone was not sufficient to shift the targeting of hFis1 to peroxisomes, further regulative mechanisms are likely to be involved. The findings indicate that targeting of hFis1 to peroxisomes and mitochondria are independent events and support a direct, Pex19p-dependent targeting of peroxisomal tail-anchored proteins. Furthermore, Pex11pβ and its isoforms Pex11pα and Pex11pγ have been studied. Pex11 proteins are the only proteins known to induce peroxisome elongation and proliferation in mammals, and it is assumed that they are key components in the regulation of peroxisome abundance. In this study, a comparative characterization of the Pex11p isoforms was performed for the first time. Differently tagged and truncated versions were generated and alterations of peroxisome formation and division were monitored. Interestingly, it was shown that the Pex11 proteins have (only) partially overlapping functions. Pex11pβ expression is known to induce formation of tubular peroxisomes, followed by segmentation of the tubules before they are divided by the division machinery. It was demonstrated here that Pex11pγ promotes tubulation of peroxisomes similar to Pex11pβ, but does not induce subsequent segmentation of the peroxisomal tubules. Pex11pα, on the other hand, induces only a segregation of peroxisomal proteins. Thus, Pex11pα and Pex11pγ appear to fulfil different functions, which are combined in Pex11pβ. Furthermore, the Pex11 proteins show different sensitivities to the detergent Triton-X 100, which is likely to be related with different lipid binding properties, which might in turn explain their capacities to deform membranes. Several signals inducing peroxisome elongation (e.g. microtubule depolymerisation) were examined, and it was demonstrated that multiple simultaneous stimuli result in hypertubulation of peroxisomes. The hypertubulation was not mediated by transcriptional upregulation of the Pex11 proteins. This additive effect indicates that complex regulation and activation mechanisms of the Pex11 proteins exist (e.g. post-translational modifications), and/or that other effectors than Pex11pβ are able to mediate elongation of peroxisomal membranes. Moreover, it was discovered that a Pex11pβ-YFP fusion protein can be used as a specific tool to further dissect the growth and division process at early time points. Pex11pβ-YFP inhibits peroxisomal segmentation and division, but instead results in formation of pre-peroxisomal membrane structures composed out of globular domains and tubular extensions. These structures were characterized in detail. Interestingly, peroxisomal matrix and membrane proteins were targeted to distinct regions of the peroxisomal structures. Performing time-course and import assays, it was shown that Pex11pβ-mediated membrane formation was initiated at the pre-existing peroxisome. This indicates that growth and division of peroxisomes follows a multistep maturation pathway and that formation of mammalian peroxisomes is more complex than simple division of a pre-existing organelle. In this study, the early steps of the peroxisomal growth and division process have been characterized in detail for the first time. The findings give new insights into the general processes of peroxisome formation by growth and division, and indicate the involvement of new, not yet characterized processes (e.g. in protein sorting) at the peroxisomal membrane.