A major unsolved problem of cell biology and biochemistry is the elucidation of the mechanisms of intracellular phospholipid transport and assembly into the organelles of eukaryotic cells. While considerable information has accrued to define the mechanisms of intracellular protein transport, little detailed information is available to describe the lipid transport at the molecular level. Lipid transport is essential for all cell growth, development, replication and homeostasis. The long term goals of this project are to define intracellular lipid transport at the molecular level using biochemical and genetic approaches in the yeast Saccharomyces cerevisiae. This proposal will extend recent advances made in identifying specific genes involved in controlling the movement of phosphatidylserine and phosphatidylethanolamine among different organelles.
In Specific Aim 1 we will address the role of protein ubiquitination in regulating phosphatidylserine transport between the endoplasmic reticulum and mitochondria. We will use biochemical and genetic manipulations in vitro and in vivo to regulate protein ubiquitination, and perform proteomic analysis to identify novel substrates for ubiquitination.
In Specific Aim 2 we will examine the role of specific lipids, proteins, and protein subdomains in promoting phosphatidylserine transport between the endoplasmic reticulum and Golgi. We will use permeabilized cells and isolated organelles derived from wild type and mutant strains to identify specific lipid and protein components required for this phosphatidylserine transport.
Specific Aim 3 will focus upon the genetics and biochemistry of phosphatidylethanolamine export from the Golgi to the endoplasmic reticulum. We will characterize the transport defects caused by multiple genes implicated in the process by previous genetic screens and develop reconstitution methods for the process in vitro. Our combined genetic and biochemical approach will provide new molecular and mechanistic information about the phospholipid transport processes in eukaryotic cells and provide new insights into the regulation of membrane biogenesis.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
5R37GM032453-24
Application #
7087661
Study Section
Physiological Chemistry Study Section (PC)
Program Officer
Chin, Jean
Project Start
1983-07-01
Project End
2008-06-30
Budget Start
2006-07-01
Budget End
2007-06-30
Support Year
24
Fiscal Year
2006
Total Cost
$357,497
Indirect Cost
Name
National Jewish Health
Department
Type
DUNS #
076443019
City
Denver
State
CO
Country
United States
Zip Code
80206
Kannan, Muthukumar; Riekhof, Wayne R; Voelker, Dennis R (2015) Transport of phosphatidylserine from the endoplasmic reticulum to the site of phosphatidylserine decarboxylase2 in yeast. Traffic 16:123-34
Riekhof, Wayne R; Naik, Surabhi; Bertrand, Helmut et al. (2014) Phosphate starvation in fungi induces the replacement of phosphatidylcholine with the phosphorus-free betaine lipid diacylglyceryl-N,N,N-trimethylhomoserine. Eukaryot Cell 13:749-57
Riekhof, Wayne R; Wu, Wen-I; Jones, Jennifer L et al. (2014) An assembly of proteins and lipid domains regulates transport of phosphatidylserine to phosphatidylserine decarboxylase 2 in Saccharomyces cerevisiae. J Biol Chem 289:5809-19
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Bobenchik, April M; Choi, Jae-Yeon; Mishra, Arunima et al. (2010) Identification of inhibitors of Plasmodium falciparum phosphoethanolamine methyltransferase using an enzyme-coupled transmethylation assay. BMC Biochem 11:4
Brechbuhl, Heather M; Gould, Neal; Kachadourian, Remy et al. (2010) Glutathione transport is a unique function of the ATP-binding cassette protein ABCG2. J Biol Chem 285:16582-7

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