Stefano, Giovanni: Trans-Golgi Network as independent organelle from Golgi apparatus in plant cells. - Bonn, 2009. - Dissertation, Rheinische Friedrich-Wilhelms-Universität Bonn.
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author = {{Giovanni Stefano}},
title = {Trans-Golgi Network as independent organelle from Golgi apparatus in plant cells},
school = {Rheinische Friedrich-Wilhelms-Universität Bonn},
year = 2009,
month = sep,

note = {In eukaryotic cells, GTPase-Activating Proteins (GAPs) are a family of proteins, which acts on small GTP-binding proteins of the Ras superfamily. GAP proteins have a conserved structure and use similar mechanisms, promoting hydrolysis of GTP to GDP. GAPs include several groups based on their substrate proteins, such as ARF (ADP Ribosylation Factor) GAPs, RAB (RAS-like protein in Brain) GAPs, and RHO (RAS Homologue) GAPs.
ARFGAPs act specifically inducing hydrolysis of GTP on ARFs. In Arabidopsis thaliana genome, there are 15 proteins with ARFGAP domains (named AGD1-15) which are classified as ARFGAP Domain (AGD) proteins. These proteins have been highly conserved during the evolution of eukaryotes.
In this thesis, the cellular role of an ARFGAP (AGD5) has been investigated. This group of proteins includes five ARFGAP members (AGD5-AGD10) which contain only the AGD domain at the amino terminus. These proteins are structurally related to the yeast ARFGAPs (Age2p, Gcs1p and Glo3p) which perform their function at the TGN (Trans-Golgi Network). Mutagenesis experiments in yeast cells that have a suppressed GAP activity for Glo3p and Gsc1p showed an impaired retrograde protein transport from ER to Golgi apparatus.
In animal cells, overexpression of ARF1GAP determines re-absorbance of Golgi membrane proteins into the ER disrupting retrograde trafficking, as shown using brefeldin-A (BFA), which is a protein trafficking inhibitor.
This suggests that ARF1GAP play a regulator role towards ARF during Golgi apparatus to ER transport.
ARFGAPs are generally considered a group of proteins, which stimulate the intrinsic GTPase activity of ARF proteins. However, an additional role has been suggested in the regulation of membrane traffic.
Thus, GAP proteins may play a crucial role in regulating the disassembly and dissociation of vesicle coats. In plant cells, as in animal and yeast cells, ARFs may also have different effectors and regulator proteins that can control the trafficking pathway. Analysis of the Arabidopsis thaliana genome has highlighted the conservation of numerous proteins including the GAP proteins. Among all these proteins, only a few have been characterized in detail. Recent studies have shown that three ARFGAPs, called VAN3, OsAGAP, SCARFACE, may play an important role in vesicle transport from the plasma membrane to the endosomes, and vice versa, having a role also in auxin signalling. In plant cells, the precise function of ARFGAPs at the TGN and its regulators are unknown.
In this thesis, the biological function of an ARFGAP, classified as AGD5, from A. thaliana has been investigated by using confocal microscopy techniques, site-directed mutagenesis and biochemical experiments. Using these methodologies, the sub-cellular localization and biological role of AGD5 in protein trafficking in plant cells were investigated.
YFP-AGD5 localizes to TGN
To determine the subcellular localization of AGD5, a protein fusion construct with YFP (yellow fluorescent protein) was produced, YFP-AGD5, and expressed in tobacco leaf epidermal cells. Laser confocal microscopy analyses indicated thatYFP-AGD5 labelled mobile punctate structures that were motile in the cell. To identify the nature of the structures, Nicotiana tabacum leaf epidermal cells were cotransformed with YFP-AGD5 fusion and various Golgi apparatus, TGN and endosome markers. It was found that the distribution of YFP-AGD5 was different compared to ERD2, which is a Golgi apparatus marker. Instead it was partly similar to that of ARF1, which mainly localize to the Golgi apparatus but also to additional non-Golgi structures. This shows that YFP-AGD5 and ARF1 co-localize to extra Golgi structures. Furthermore, YFP-AGD5 was coexpressed with various endocytic and TGN markers and it was found that AGD5 labels compartments stained with SYP61, which is also a TGN marker.
YFP- AGD5[R59Q] localize to TGN, and functions on ARF1 in vivo
It has been shown that YFP-AGD5 colocalizes with ARF1 on the TGN. Judging from its subcellular localization, AGD5 probably acts as an ARFGAP on ARF1. It was examined whether AGD5 acts on ARF1 in vivo by coexpressing tobacco cells with YFP tagged AGD5[R59Q] a GAP-negative mutant.
The cells expressing ARF1 alone showed a punctate subcellular distribution, which represents Golgi apparatus and non-Golgi structures. In contrast, in cells coexpressing ARF1 and AGD5[R59Q], ARF1 was distributed at punctate structures and in the cytosol. This suggests that ARF1 in such cells may remain as a GTP-bound form on membranes where AGD5 is normally the primary ARFGAP. In any case, the above observations indicate that AGD5 is likely to function in an ARF1 dependent process.
AGD5 interacts with ARF1 in vivo and in vitro
The results of the previous section indirectly suggest that AGD5 is involved in the activation of ARF1 on the TGN in plants. To confirm the role of AGD5 in the activation of ARF1 to the TGN, and to obtain direct evidence that AGD5 would alter ARF1 distribution a glutathione agarose affinity assay based on the interaction of a recombinant GST-AGD5 with mutant GTP bound form ARF1-YFP protein expressed in tobacco leaves was developed.
Results indicate that there is an interaction between AGD5 and ARF1GTP. However, the data do not allow us to establish whether the binding of ARF1 to AGD5 is direct. Therefore, to determine if the interaction between ARF1 and AGD5 required the presence of other cytosolic or TGN associated proteins, ARF1 and AGD5 were produced in E. coli and tested for the interaction with ARF1 and purified AGD5 in vitro. This experiment demonstrates that the interaction of ARF1 with AGD5 is not dependent on other cytosolic proteins and it could be due to a direct association of the two molecules.
Overexpression of YFP-AGD5 in Arabidopsis stable plants
To determine the cellular expression pattern of AGD5 in plants, the P35S:-YFP-AGD5 construct was introduced into A. thaliana via Agrobacterium-mediated transformation. Confocal microscopy on transgenic lines showed that the fluorescence is mainly distributed in the root. The subcellular distribution of YFP-AGD5 was detected as punctate structures along the root but mainly in the apical part. Analysis of root hairs in transgenic lines overexpressing AGD5 displays the bulged root hair phenotype. Furthermore, the overexpression causes defects in root tip growth.
Additionally, AGD5 in pollen of transgenic Arabidopsis plants was found to cause various pollen tube phenotypes, including expanded tubes with swollen tips, twisted tubes, and bifurcate tips.
AGD5 affects protein secretion in N. tabacum transformed protoplasts
To demonstrate further that AGD5[R59Q] (GAP-negative mutant) has a negative effect compared to AGD5 wild type form on protein export from the TGN, tobacco leaf protoplasts were cotransfected with the secretory marker SecRGUS. AGD5 did not affect SecRGUS secretion, but the AGD5[R59Q] mutant exhibited a negative effect on the secretion of SecRGUS. These data mirror our confocal microscopy results showing that AGD5 affected the distribution of ARF1 at the TGN, suggesting that the mutant form may block anterograde (from ER to Golgi apparatus) export.
This work established that AGD5 localizes to the TGN. Furthermore, this study has highlighted a new interactor, an ARFGAP, for the small GTPase ARF1 protein at the TGN organelle suggesting an additional role in vesicle transport along the endocytic pathway. Therefore, this work represents a starting point to analyze the AGD5 influence on ARF1 functionality during auxin receptor recycling.},

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