Dissertation (Ph.D.) -- University of Texas Southwestern Medical Center at Dallas, 2006. / Vita. Bibliography: p. 87-94.

In Saccharomyces cerevisiae, experiments with temperature-sensitive mutants of the PI4-kinase Pik1p revealed that the PI4P pool generated by this enzyme is essential for Golgi morphology and normal secretory function and that the PI4P pool at the Golgi represents a regulatory signal on its own. In order to function as a spatial and temporal regulator of membrane traffic, PI4P synthesis and turnover must be tightly regulated. It remains elusive which factors are involved in the targeting and regulation of Pik1p. Little is also known about PI4P binding proteins mediating the effects of this phosphoinositide on Golgi function. Since it has been shown that multiple pathways leave the Golgi towards the plasma membrane one can ask the question whether Pik1p and its product PI4P specifically control one pathway? Here we demonstrate an interaction of Pik1p with the 14-3-3 proteins Bmh1p and Bmh2p. Interestingly, overexpression of Bmh1p and Bmh2p results in multiple genetic interactions with genes involved in late steps of exocytosis and it affects the forward transport of the general amino acid permease Gap1p. The detected interaction depends on the phosphorylation state of Pik1p and Pik1p phosphorylation accompanies its shuttling out of the nucleus into the cytoplasm where presumably the binding to Bmh1/2p occurs. Therefore, we reason that these interactions might serve the sequestration of Pik1p away from the Golgi. This study reveals that Pik1p shows a strong effect on the delivery of Gap1p to the surface whereas the transport of exocytosis markers implicated in the direct Golgi-to-plasma membrane pathway are not significantly disturbed. Cells carrying a deletion of gga2 also show a strong defect in delivery of Gap1p to the surface. In addition, pik1-101 gga2[delta]double mutants display synthetic genetic and membrane transport phenotypes and recruitment of Gga2 to the TGN partially depends on functional Pik1p. Therefore, our results suggest a role of Pik1p in the TGN to endosome pathway.

The lipid kinase phosphatidylinositol 4-kinase III β (PI4KIIIβ) phosphorylates phosphatidylinositol (PtdIns) to generate PI(4)P in the Golgi. PI4KIIIβ is likely involved in the development of breast cancer as it has been reported genetically amplified in a subset of human breast tumours and is a downstream effector of the eukaryotic elongation factor 1 alpha 2 (eEF1A2), a transforming gene that is amplified and highly expressed in approximately 60% of human breast tumours. The goal of my thesis is to investigate a role for PI4KIIIβ in breast oncogenesis. We show that PI4KIIIβ is highly expressed in approximately 20% of primary human breast tumours. Overexpression of PI4KIIIβ in an invasive breast ductal carcinomas cell line, BT549, increased the production of filopodial actin filament protrusions and enhanced in vitro proliferative capacity. Enhanced PI4KIIIβ expression did not impact the migratory rate of these breast cancer cells. We found that PI4KIIIβ expression activates Akt kinase in the BT549 breast cancer cell line. PI4KIIIβ overexpression led to an increase in the plasma membrane abundance of the PI3K derived PI(3,4,5)P3/PI(3,4)P2 lipids, upstream activators of Akt signalling. PI(4)P and PI(4,5)P2 are precursors to PI(3,4,5)P3 and PI(3,4)P2 generation, however, no changes in the overall cellular abundance or localization of PI(4)P or PI(4,5)P2 were detected in PI4KIIIβ-overexpressing cells. Inhibition of PI4KIIIβ kinase activity, using the drug Pik93, had no effect on PI4KIIIβ-mediated Akt activation. Additionally, ectopic expression of a catalytically inactive PI4KIIIβ also led to increased Akt activity and PI(3,4,5)P3/PI(3,4)P2 plasma membrane abundance. Together, this implies that PI4KIIIβ regulates Akt independently of PI(4)P generation. The PI4KIIIβ interacting protein, Rab11, is likely involved in PI4KIIIβ mediated Akt activation, as RNAi-mediated depletion of Rab11 suppressed the effect of PI4KIIIβ overexpression on Akt activation. Furthermore, PI4KIIIβ overexpression altered cellular Rab11 distribution and led to enhanced recruitment of PI4KIIIβ and Rab11 to recycling endosomes. Therefore, PI4KIIIβ is highly expressed in a subset of breast tumours and upregulated PI4KIIIβ expression enhances filopodia production and cell growth in vitro. Enhanced PI4KIIIβ expression increases PI(3,4,5)P3/PI(3,4)P2 plasma membrane abundance and Akt activation independently of its kinase function, through a mechanism that likely involves Rab11. This work suggests that PI4KIIIβ impacts breast oncogenesis by regulating PI3K/Akt signalling through Rab11 and endosomal trafficking.

Chez les plantes, l'acide salicylique (SA) a un rôle central dans la réponse à de nombreuses contraintes environnementales et lors du développement. Cependant les événements de signalisation précoces qu'il déclenche sont peu connus. Nous montrons, par marquage métabolique au 33Pi sur une suspension cellulaire d'Arabidopsis thaliana, que le SA induit une diminution rapide et précoce d'un pool de phosphatidylinositol (PI). Celle-ci est accompagnée d'une accumulation de PI 4-phosphate et PI 4,5-bisphosphate. Ces changements sont inhibés par de la wortmannine à 30 μM mais pas à 1 μM, ce qui implique une activation de PI 4-kinase de type III. C'est pourquoi une étude des effets de la wortmannine sur les modifications de transcriptome par le SA a été menée à l'aide de la puce " Complete Arabidopsis Transcriptome MicroArray " (CATMA). Sur 773 gènes régulés par le SA, 112 sont sensibles à 30 μM de wortmannine. En parallèle, nous voyons que l'acide phosphatidique issu de la phospholipase D (PLD) est important pour la réponse génique précoce au SA. Une expérience de puces menée pour identifier les gènes régulés par la PLD en réponse au SA a révélé que parmi 1327 gènes régulés par le SA, 97 gènes sont régulés positivement, et 117 gènes négativement, par la PLD. Les régulons de la voie sensible à la wortmannine et de la voie PLD se chevauchent fortement, ce qui suggère que les deux activités agissent en synergie dans la même voie de signalisation en réponse au SA.

Regulated secretion of hormones, digestive enzymes and other biologically active molecules requires formation of secretory granules. However, the molecular machinery required for secretory granule biogenesis is incompletely understood. I used powerful genetic approaches available in the fruit fly Drosophila melanogaster to investigate the factors required for biogenesis of mucin-containing ‘glue granules,’ which form within epithelial cells of the third-instar larval salivary gland. I discovered that clathrin and the clathrin adaptor protein complex (AP-1), as well the enzyme type II phosphatidylinositol 4-kinase (PI4KII), are indispensable for glue granule biogenesis. Clathrin and AP-1 are necessary for maturation of exocrine, endocrine and neuroendocrine secretory granules in mammalian cells. I found that Drosophila clathrin and AP-1 colocalize at the TGN and that clathrin recruitment requires AP-1. I further showed that clathrin and AP-1 colocalize with secretory cargo at the TGN and on glue granules. Finally, I demonstrated that loss of clathrin or AP-1 leads to a profound block in secretory granule biogenesis. These findings establish a novel role for AP-1/clathrin-dependent trafficking in the formation of mucin-containing secretory granules. Type II phosphatidylinositol 4-kinase (PI4KII) generates the membrane lipid phosphatidylinositol 4-phosphate (PI4P) at the trans-Golgi network and is required to recruit cargo to endosomes in mammalian cells. I generated null mutations in the sole Drosophila PI4KII and demonstrated a role for PI4KII in both glue granule and pigment granule biogenesis. PI4KII mutant salivary gland cells exhibit small glue granules and mislocalize glue protein to abnormally large late endosomes. Additionally, PI4KII mutants exhibit altered distribution of the granule specific SNARE, SNAP-24. These data point to a crucial role for PI4KII in sorting of regulated secretory products during granule biogenesis. Together, my results indicate that the larval salivary gland is a valuable system for investigating molecular mechanisms involved in secretory granule biogenesis, and provide a framework for future studies using this system.

Regulated secretion of hormones, digestive enzymes and other biologically active molecules requires formation of secretory granules. However, the molecular machinery required for secretory granule biogenesis is incompletely understood. I used powerful genetic approaches available in the fruit fly Drosophila melanogaster to investigate the factors required for biogenesis of mucin-containing ‘glue granules,’ which form within epithelial cells of the third-instar larval salivary gland. I discovered that clathrin and the clathrin adaptor protein complex (AP-1), as well the enzyme type II phosphatidylinositol 4-kinase (PI4KII), are indispensable for glue granule biogenesis. Clathrin and AP-1 are necessary for maturation of exocrine, endocrine and neuroendocrine secretory granules in mammalian cells. I found that Drosophila clathrin and AP-1 colocalize at the TGN and that clathrin recruitment requires AP-1. I further showed that clathrin and AP-1 colocalize with secretory cargo at the TGN and on glue granules. Finally, I demonstrated that loss of clathrin or AP-1 leads to a profound block in secretory granule biogenesis. These findings establish a novel role for AP-1/clathrin-dependent trafficking in the formation of mucin-containing secretory granules. Type II phosphatidylinositol 4-kinase (PI4KII) generates the membrane lipid phosphatidylinositol 4-phosphate (PI4P) at the trans-Golgi network and is required to recruit cargo to endosomes in mammalian cells. I generated null mutations in the sole Drosophila PI4KII and demonstrated a role for PI4KII in both glue granule and pigment granule biogenesis. PI4KII mutant salivary gland cells exhibit small glue granules and mislocalize glue protein to abnormally large late endosomes. Additionally, PI4KII mutants exhibit altered distribution of the granule specific SNARE, SNAP-24. These data point to a crucial role for PI4KII in sorting of regulated secretory products during granule biogenesis. Together, my results indicate that the larval salivary gland is a valuable system for investigating molecular mechanisms involved in secretory granule biogenesis, and provide a framework for future studies using this system.

In Saccharomyces cerevisiae, experiments with temperature-sensitive mutants of the PI4-kinase Pik1p revealed that the PI4P pool generated by this enzyme is essential for Golgi morphology and normal secretory function and that the PI4P pool at the Golgi represents a regulatory signal on its own. In order to function as a spatial and temporal regulator of membrane traffic, PI4P synthesis and turnover must be tightly regulated. It remains elusive which factors are involved in the targeting and regulation of Pik1p. Little is also known about PI4P binding proteins mediating the effects of this phosphoinositide on Golgi function. Since it has been shown that multiple pathways leave the Golgi towards the plasma membrane one can ask the question whether Pik1p and its product PI4P specifically control one pathway? Here we demonstrate an interaction of Pik1p with the 14-3-3 proteins Bmh1p and Bmh2p. Interestingly, overexpression of Bmh1p and Bmh2p results in multiple genetic interactions with genes involved in late steps of exocytosis and it affects the forward transport of the general amino acid permease Gap1p. The detected interaction depends on the phosphorylation state of Pik1p and Pik1p phosphorylation accompanies its shuttling out of the nucleus into the cytoplasm where presumably the binding to Bmh1/2p occurs. Therefore, we reason that these interactions might serve the sequestration of Pik1p away from the Golgi. This study reveals that Pik1p shows a strong effect on the delivery of Gap1p to the surface whereas the transport of exocytosis markers implicated in the direct Golgi-to-plasma membrane pathway are not significantly disturbed. Cells carrying a deletion of gga2 also show a strong defect in delivery of Gap1p to the surface. In addition, pik1-101 gga2[delta]double mutants display synthetic genetic and membrane transport phenotypes and recruitment of Gga2 to the TGN partially depends on functional Pik1p. Therefore, our results suggest a role of Pik1p in the TGN to endosome pathway.

Over the past few years a large amount of work has provided growing insight into the molecular mechanisms that direct post-Golgi trafficking events in the budding yeast Saccharomyces cerevisae. However, a key event in this process, the formation of secretory vesicles at the Golgi and sorting of cargo into these transport carriers, remains poorly understood. It has been demonstrated that phosphatidylinositol 4-phosphate (PI(4)P) generated by the PI(4)-kinase Pik1p plays an essential role in maintenance of Golgi secretory function and morphology. Up to now relatively few targets of Pik1/PI(4)P signaling at the Golgi have been identified and it thus remains elusive how Pik1p mediates its essential function in Golgi secretion. During my thesis work, I used synthetic genetic array analysis (SGA) of a temperature-sensitive mutant allele of PIK1 (pik1-101) in order to gain better understanding of Pik1p function at the TGN and to isolate new regulators of post-Golgi transport in yeast. I identified a total of 85 genes, whose deletion resulted in a synthetic growth defect when combined with the pik1-101 mutation. 21 isolated deletion mutants were used for further analysis, several of which were found to share common trafficking phenotypes with the pik mutant. A striking result of the screen was the finding that Pik1p interacts genetically with several components of a potential post-translational modification pathway referred to as “urmylation pathway”. In addition, a novel, previously uncharacterized subunit of the Transport protein particle (TRAPP) complex was isolated as genetic interactor of Pik1p, suggesting a function for the TRAPP complex in a Pik1p dependent trafficking pathway. Using tandem affinity purification, I could also demonstrate that TRAPP shows previously unknown interactions with other regulators of post-Golgi transport. The second part of this thesis describes the development of a new visual screening approach. Recent work indicates that secretory cargo in yeast can be transported to the cell surface via at least two different exocytic branches. Upon block of one pathway cargo can be partially redistributed into the other pathway. This partial redundancy of exocytic pathways provides one explanation why genetic screens in the past were largely unsuccessful in identifying the molecular machinery that directs vesicle budding and cargo sorting at the TGN. I collaborated in the development of a novel screening method that was devised to circumvent this problem. The method took advantage of the systematic yeast knockout array and was based on the assumption that a defect in cargo sorting and cell surface transport could be detected as intracellular accumulation of a GFP-tagged model cargo. The suitability of our approach for identifying regulators of secretory transport has been demonstrated in a small-scale pilot study that will be presented in this thesis. The screening method proofed to be applicable on a genome-wide scale and can now be used for the screening of additional markers. This novel approach provides an entry point to the comprehensive study of TGN sorting.

Over the past few years a large amount of work has provided growing insight into the molecular mechanisms that direct post-Golgi trafficking events in the budding yeast Saccharomyces cerevisae. However, a key event in this process, the formation of secretory vesicles at the Golgi and sorting of cargo into these transport carriers, remains poorly understood. It has been demonstrated that phosphatidylinositol 4-phosphate (PI(4)P) generated by the PI(4)-kinase Pik1p plays an essential role in maintenance of Golgi secretory function and morphology. Up to now relatively few targets of Pik1/PI(4)P signaling at the Golgi have been identified and it thus remains elusive how Pik1p mediates its essential function in Golgi secretion. During my thesis work, I used synthetic genetic array analysis (SGA) of a temperature-sensitive mutant allele of PIK1 (pik1-101) in order to gain better understanding of Pik1p function at the TGN and to isolate new regulators of post-Golgi transport in yeast. I identified a total of 85 genes, whose deletion resulted in a synthetic growth defect when combined with the pik1-101 mutation. 21 isolated deletion mutants were used for further analysis, several of which were found to share common trafficking phenotypes with the pik mutant. A striking result of the screen was the finding that Pik1p interacts genetically with several components of a potential post-translational modification pathway referred to as “urmylation pathway”. In addition, a novel, previously uncharacterized subunit of the Transport protein particle (TRAPP) complex was isolated as genetic interactor of Pik1p, suggesting a function for the TRAPP complex in a Pik1p dependent trafficking pathway. Using tandem affinity purification, I could also demonstrate that TRAPP shows previously unknown interactions with other regulators of post-Golgi transport. The second part of this thesis describes the development of a new visual screening approach. Recent work indicates that secretory cargo in yeast can be transported to the cell surface via at least two different exocytic branches. Upon block of one pathway cargo can be partially redistributed into the other pathway. This partial redundancy of exocytic pathways provides one explanation why genetic screens in the past were largely unsuccessful in identifying the molecular machinery that directs vesicle budding and cargo sorting at the TGN. I collaborated in the development of a novel screening method that was devised to circumvent this problem. The method took advantage of the systematic yeast knockout array and was based on the assumption that a defect in cargo sorting and cell surface transport could be detected as intracellular accumulation of a GFP-tagged model cargo. The suitability of our approach for identifying regulators of secretory transport has been demonstrated in a small-scale pilot study that will be presented in this thesis. The screening method proofed to be applicable on a genome-wide scale and can now be used for the screening of additional markers. This novel approach provides an entry point to the comprehensive study of TGN sorting.