Phosphoinositide Signaling and Polarized Membrane Growth: A Perspective from Sec14-Like PITPs

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Dokumentart: PhDThesis
Date: 2015-06-30
Language: English
Faculty: 7 Mathematisch-Naturwissenschaftliche Fakultät
Department: Biologie
Advisor: Schaaf, Gabriel (Dr.)
Day of Oral Examination: 2014-06-17
DDC Classifikation: 570 - Life sciences; biology
Keywords: Wurzelhaar
Other Keywords: Plasmamembran-Assoziirungsmodul
Plasma membrane association
Sec14 proteins
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Sec14-like phosphatidylinositol/phosphatidylcholine (PtdIns/PtdCho) transfer proteins (PITPs) represent important regulatory components that integrate phospholipid metabolism and membrane trafficking in eukaryotes. Data derived from yeast studies suggest that this regulation results from a Sec14-mediated PtdIns/PtdCho exchange reaction that stimulates PtdIns kinase activity, thus facilitating the generation of phosphoinositides (PIPs) such as PtdIns(4)P and PtdIns(4,5)P2. The present work reports recent findings on the roles and functional mechanisms of yeast and plant Sec14-like PITPs. The Arabidopsis Sec14-nodulin AtSFH1, an important regulator of root hair formation, was used as a model to show that the nodulin domain represents a plasma membrane association module with high binding specificity towards PtdIns(4,5)P2. A Lys-rich C-terminal motif is necessary for PIP binding activity, which is amplified by homo-oligomerization of the nodulin domain. Both PIP association and homooligomerization are essential properties of AtSFH1 and mutants defective in PtdIns(4,5)P2 binding can be rescued by a translational fusion with a bona fide PtdIns(4,5)P2 binding domain. A model for the mode of action of AtSFH1 is proposed herein, and suggests that the physical linkage of the N-terminal Sec14 and the nodulin domains couples PIP synthesis and organization, so as to promote defined landmarks for PIP effectors that modulate developmental control of polarized membrane growth. A second approach used to gain further insights on AtSFH1 functions is also discussed. EMS-mutagenized plants that suppress the atsfh1-1-dependent short root hair henotype were isolated. By employing nextgeneration sequencing combined with deep candidate resequencing (dCARE), a mutation in the AtSTR1 sulfurtranferase encoding gene was identified. The preliminary results presented here suggest that cyanide detoxifying enzymes could represent additional cellular components that regulate root hair development, possibly in a pathway that is independent of AtSFH1. Finally, a directed evolution screen was performed in order to identify mutations that confer Sec14-like activities to the functionally inactive yeast Sec14 homolog Sfh1. Biochemical, biophysical and computational approaches helped to discover a network of transient interactions that propagates conformational energy from the lipid binding pocket to the ‘helical gating module’ that controls lipid access, thus enhancing the rates of phospholipid exchange and presentation in the mutant proteins. Taken together, the discoveries presented herein provide important details into the mechanisms regulating phospholipid exchange and PIP organization by Sec14-like PITPs, as well as comprehensive clues of how these fascinating proteins ultimately promote phosphoinositide homeostasis in eukaryotes.

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