Polarized hyphal growth dominates the life cycle of filamentous fungi and is essential to disease progression for many fungal pathogens. Despite its importance, much of the basic biology controlling the process remains to be elucidated. Protein Nmyristoylation is one process important to hyphal growth for which the direct mechanism for this connection is not understood. N-myristoylation is mediated by Nmyristoyltransferase (NMT), which links 14-carbon myristate to target proteins. In Aspergillus nidulans, a mutation in the NMT gene (swoF1) results in abnormal morphogenesis during spore germination and the establishment of hyphal polarity. I hypothesize that a protein or proteins downstream of NMT are important for polarized hyphal growth. Using a forward genetic approach, I obtained six suppressors of swoF1. I found that three were proteasome-related and a mutation in genes encoding 26S proteasome subunits by-passed the polarity defects of swoF1. Interestingly, N-myristoylation negatively regulated the activity of the 26S proteasome. This result was confirmed by treating with the proteasome inhibitor MG132. This is the first finding of a connection between N-myristoylation and proteasome function during polarized growth. To identify targets by reverse genetic analysis, I found that 41 proteins (of more that 10,000 encoded by the organism) were predicted to be myristoylated in silico. Three were ADP ribosylation factors (ARF), proteins known to be involved in vesicle formation and trafficking in other systems. I chose ArfA (AN1126.3), ArfB (AN5020.3), and ArlA (AN5912.3) for further characterization of polarization in this study. ArfA::GFP discretely localized to endomembrane likely to be Golgi bodies. ArfB::GFP localized to septa and plasma membrane. N-myristoylation determined the localization of both ArfA and ArfB. Disruption of the arfB gene resulted in loss of polarity establishment and endocytosis. Together these results suggest that endocytosis plays an important role in maintaining hyphal polarized growth and in shaping the cell apex.

The endosomal trafficking system is a network of highly coordinated cellular pathways that control the growth and function of cells. The coordination of secretion and endocytosis in cells is one of the primary drivers of polarized growth, where new plasma membrane and cell wall components are deposited at the growing apex. In plants, one of the cell types exhibiting polarized growth are the root hairs. Root hairs are regulated extensions of epidermal cells called trichoblasts and are essential for anchorage, absorption of water and nutrients, and plant-microbe interactions. In this thesis, I characterize a previously undescribed protein involved in retromer function and endosomal trafficking pathways that regulate tip growth in root hairs of Arabidopsis thaliana. The large retromer complex functions in recycling receptors in endosomal trafficking pathways essential for diverse developmental programs including cell polarity, programmed cell death, and shoot gravitropism in the model plant, Arabidopsis thaliana. I have characterized VPS26C, a novel member of the large retromer complex, that is essential in maintaining root hair growth in Arabidopsis. We used Bimolecular Fluorescence Complementation (BiFC) analysis to demonstrate thatVPS26C interacts with previously characterized core retromer subunits VPS35A and VPS29. Genetic analysis also indicates that vps26c suppresses the root hair growth and cell wall organization phenotypes of a null mutant of the SNARE VTI13 that localizes to early endosomes and the vacuole membrane, indicating a crosstalk between the VPS26C-retromer and VTI13-dependent vesicular trafficking pathways. Phylogenetic analysis was used to show that VPS26C genes are present in most angiosperms but appear to be absent in monocot genomes. Moreover, using a genetic complementation assay, we have demonstrated that VPS26C shares deep conservation of biochemical function with its human ortholog (DSCR3/VPS26C). We also used an affinity purification-based proteomic analysis to identify proteins associated with VTI13 in young seedlings. Preliminary results suggest that a number of proteins linked to cell plate organization in plants are associated with the VTI13 proteome, emphasizing the potential role of this pathway in new cell wall biosynthesis/organization. Additionally, we have identified endoplasmic reticulum (ER)-body proteins, involved in plant defense response pathways, suggesting that either the VTI13 endosomal trafficking pathway is functioning in plant defense responses, or the ER-body proteins have additional independent function(s) in Arabidopsis roots that depend on VTI13. In summary, I have described a novel retromer complex essential for polarized growth in Arabidopsis. VPS26C is an ancient gene and shares sequence and functional homology between human and Arabidopsis. vps26c is a genetic suppressor of the vti13- dependent root hair growth and cell wall organization pathways. Proteomic analysis of VTI13 endosomes in young seedlings suggests that a number of proteins associated with cell plate formation are associated with VTI13 compartments, supporting the genetic analysis described here and serves as a starting point to further describe the role of this pathway in controlling polarized growth in plants.

Les phosphoinositides sont des molécules régulatrices présentes à l'interface membrane-cytosol, impliquées dans la transduction du signal, le trafic membranaire ainsi que l'organisation du cytosquelette. Ces lipides recrutent non seulement diverses protéines vers des compartiments spécifiques, mais régulent aussi leur activité enzymatique. Chez la levure Saccharomyces cerevisiae, ils interagissent directement avec le domaine RhoGAP de la protéine Rgd1, identifiée comme un activateur commun aux GTPases Rho3 et Rho4. Ces 2 protéines, respectivement impliquées dans la croissance polarisée et la cytocinèse, voient leur activité GTPasique exacerbée en présence de Rgd1pet des PIPs. L'objectif de cette thèse était comprendre à l'échelle moléculaire le processus unique d'activation de RhoGAP par les PIPs. Pour ce faire, nous avons réalisé l'étude structurale de RhoGAP par cristallographie couplée à la RMN en solution. Nos résultats montrent que le domaine possède les éléments essentiels à l'activation des protéines Rho. L'interaction avec les PIPs a été suivie par RMN en présence de PI(4)P et de PI(4,5)P2, respectivement localisés dans les vésicules de sécrétion et à la membrane plasmique. Nos résultats révèlent un site de liaison commun aux PIPs dans une région non conservée chez les domaines RhoGAP. L'affinité des complexes, de l'ordre de la centaine de micromolaires suggèrent qu'in vivo l'interaction soit transitoire et réversible avec les PIPs. La sélectivité de l'interaction se ferait donc de façon spatio-temporelle, au niveau des vésicules de sécrétion pour la croissance polarisée et de la membrane plasmique pour la cytocinèse. / Phosphoinositides act as regulatory and signalling molecules at the membrane-cytosol interface in signal transduction, membrane traffic and cytoskeleton organization. These lipids recruit several proteins to specific compartments, but also regulate their activity. In the yeast Saccharomycescerevisiae, they directly bind the Rgd1-RhoGAP domain, that stimulates the GTPase activity of bothRho3p and Rho4p. The GTPase activity of these two Rho proteins, respectively involved in the polarized growth and cytokinesis of the yeast, is enhanced with the presence of Rgd1p and PIPs. The main objective of this thesis is to understand the PIP-RhoGAP interaction at the molecular level. In order to do that, we coupled X-ray structure determination to solution NMR spectroscopy on the isolated RhoGAP domain. Our results show that the domain contains the conserved elements that would usually confer the catalytic GTPase activation. We us e liquid-state NMR spectroscopy to follow the interaction with PI(4)P and PI(4,5)P2, respectively found in secretion vesicles and the plasma membrane. Our study reveals a common binding site for both PIPs in a non-conserved region in the RhoGAP domain family. We measured sub-millimolar binding affinity for PIPs. Such moderate binding affinities are consistent with the biological requirement for reversible complex formation. The selectivity of the interaction could be made in a spatio temporal way, on the secretion vesicles during polarized growth and at the plasma membrane during cytokinesis.