Substrate dependent regulation of Drosophila F -box protein

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The Drosophila embryo consists of a series of similar repeating units which provides the foundation for the subsequent development of the adult fly. This pattern of the reiterated units is established by segmentation genes which are expressed in a hierarchical regulatory cascade. We have been interested in elucidating the function of the segmentation gene, paired (prd). Paired occupies an intermediate position in the segmentation cascade and works in concert with other pair-rule genes to spatially refine the expression of genes in the lower tier of the cascade. We identified a novel Drosophila gene, partner of paired (ppa) as an inter actor of prd in a yeast two hybrid screen. The partner of paired mRNA is expressed in stripes complementary to Paired stripes. The partner of paired gene encodes an F-box protein and is a component of the protein degradation machinery. In order to further analyze Partner of paired expression and to elucidate its role in the segmentation cascade we generated an anti-Ppa antibody. Ppa protein expression is unlike the expression of its mRNA. In my thesis I have attempted to elucidate the expression pattern of Partner of paired (Ppa) and also to understand its regulation.

In the first chapter I will describe early Drosophila development leading to the formation of metameric segments and delineate the role of segmentation genes in the formation of this pattern. Since the focus of my thesis is elucidation of expression and function of Partner of paired, an F-box protein involved in protein degradation, I also provide an overview of protein degradation and its role in development in the first chapter. In the second chapter I discuss the methodology involved in the production of purified Ppa protein employed for the generation of anti-Ppa antibody. I also discuss the initial analysis of Ppa protein expression.

The third chapter presents my analysis of the regulation of Partner of paired. F-box proteins interact specifically with their substrates, and link them to the rest of the protein degradation machinery. Each F-box protein targets multiple substrates for degradation, and each organism has multiple F-box proteins that share a common degradation machine. Therefore understanding the regulation of F-box proteins will aid in understanding how cells regulate the degradation of specific substrates precisely. I have examined the regulation of Partner of paired using protein expression studies. My protein expression studies revealed that the Ppa protein is up-regulated in response to elevated levels of its substrates. My protein synthesis inhibition study using cycloheximide provides strong evidence indicating that the regulation of Ppa is controlled through protein stabilization. This provides an efficient mechanism for rapidly increasing the levels of an F-box protein in response to substrates requiring degradation. In the absence of substrates, there is high turnover of the F-box protein leading to reduced expression in a context where its function is not needed.

Our analysis of Partner of paired has led to the identification of the pair-rule segmentation gene, even-skipped as another substrate of Partner of paired. In the third chapter I describe strong evidence that suggest a role for Ppa in the regulation of Even-skipped protein levels. 6 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.