Diverse roles of phosphatidylinositol transfer protein PITPbeta in eukaryotic cells.

摘要

Phosphatidylinositol transfer proteins (PITPs) provide a dynamic interface between membrane dynamics and lipid signaling. PITP and PITPbeta encode for two soluble class I, metazoan specific PITPs. However, despite the fact that PITPalpha and PITPbeta share 77 and 95% primary sequence identity and similarity (respectively), PITPalpha and PITPbeta are functionally distinct and play nonredundant functions in cells. Whereas PITPalpha knockout mice have been created, and these mice display neuronal, intestinal, and liver pathologies, PITPbeta functionality remains largely unknown.;In this work, I addressed the distinguishing properties of PITPbeta and its role in mammalian and zebrafish cells. We identified regions of PITPbeta that contributed to its unique properties: Golgi localization and sphingomyelin (SM) binding. Two single point mutations are sufficient to ablate the SM binding properties of PITPbeta, and the Golgi localization was attributed to the combination of two regions of the protein. Next, siRNA and morpholino analyses, in mammalian cells and zebrafish respectively, were developed to understand the physiological role of PITPbeta in cells. In mammalian cells, PITPbeta was essential for cell survival. Particularly, PITPbeta-depleted cells displayed disorganized Golgi networks and anterograde trafficking defects. Surprisingly, PITPbeta was not required for cell survival or early development in zebrafish. Instead, PITPbeta maintains the outer segment of the double cone cells of the eye -- a cell type required for detection of red and green light. The lack of a housekeeping role for PITPbeta led us to examine the roles of other PITPs in zebrafish. A novel third soluble PITP in zebrafish, PITPgamma, was identified, though its function is not yet determined because we have no means of detecting the protein levels to ascertain knockdown. PITPalpha, however, had an unexpected role in early development: reduction of PITPalpha led to an arrest at an early stage of development (about 12 hpf), ultimately leading to embryonic lethality. Together, this dissertation presents novel roles for class I PITPs in zebrafish, and represents the first functional characterization of PITPbeta.