Document Type

Dissertation

Date of Degree

2008

Degree Name

PhD (Doctor of Philosophy)

Degree In

Biochemistry

First Advisor

Lois S. Weisman

Abstract

Phosphatidylinositol 3,5-bisphosphate [PI(3,5)P2] is a low-abundance signaling lipid important for the maintenance of the endomembrane system and selected membrane trafficking pathways. In yeast, in response to hyperosmotic stress, PI(3,5)P2 levels rise more than 20-fold in 5 minutes, and return to near basal levels in 30 minutes. This transient change suggests that PI(3,5)P2 levels are tightly regulated and may be involved in signaling a response to stress. In yeast, PI(3,5)P2 is synthesized through phosphorylation of PI(3)P by the PI(3)P 5-kinase Fab1. Loss of PI(3,5)P2 in yeast causes swollen vacuoles, defective retrograde trafficking from the vacuole, defective vacuole acidification, and mis-localization of a subset of vacuole lumenal proteins.

In yeast, Vac14 is a regulator of PI(3,5)P2 levels. Mammalian Vac14 and Fab1 are found in the same complex. To study the physiological significance of PI(3,5)P2, a mouse strain was generated with the Vac14 gene disrupted by a gene-trap genomic insertion. Vac14 protein was not detectable in mutant mice. In fibroblasts cultured from the mutant mice, PI(3,5)P2 and PI(5)P are decreased to 42% and 44% of the corresponding wild-type levels, respectively. The mutant mouse brains exhibit spongiform-like morphology. Cytoplasmic vacuoles are found in neuronal cell bodies of the olfactory bulb, trigeminal ganglion, and dorsal root ganglion. Non-neural tissues appear largely normal. Similar vacuoles are also found in cultured neurons and fibroblasts. In fibroblasts, these vacuoles are formed from swelling of late endosomes/lysosomes. Some early endosomes are also enlarged. A population of cation-independent mannose-6-phosphate receptor (CI-M6PR), which recycles between endosomes and the trans-Golgi network (TGN), is trapped in early and late endosomes, indicating a block in endosome-to-TGN trafficking.

These results suggest that: 1) Neurons are acutely sensitive to loss of PI(3,5)P2. 2) In mammals, PI(3,5)P2 is required for the morphology of late endosomes/lysosomes and retrograde trafficking from endosomes to the TGN. The first conclusion is supported by another mouse strain with a retro-transposon inserted in the Fig4 gene. Fig4 is another regulator of PI(3,5)P2 levels. Similar neurodegeneration was observed in the Fig4 mutant mice.

Keywords

Fab1, Vac14, PI(3, 5)P2, CMT, neurodegeneration

Pages

ix, 119 pages

Bibliography

Includes bibliographical references (pages 109-119).

Comments

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Copyright

Copyright 2008 Yanling Zhang

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