The long term goal of this research is to determine the molecular basis of membrane traffic in mammalian cells. The focus is on mannose 6-phosphate receptors (MPRs) that deliver newly synthesized lysosomal enzymes from the Golgi to pre-lysosomes, and then return to the Golgi to pick up more cargo. Several recently discovered proteins are needed for MPR transport from late endosomes to the trans Golgi network: a cargo selection protein that recognizes the MPRs in late endosomes (TIP47), a pathway- specific SNARE complex for fusion of MPR-vesicles at the TGN, and two proteins that function in vesicle tethering at the Golgi (GCC185 and RhoBTB3). The goals of this application are (1) to define precisely, the distinct routes taken by cargoes that are transported from early endosomes back to the Golgi, with focus on MPRs in comparison with cholera toxin;(2) to carry out a genome-wide, automated siRNA screen for proteins needed for MPR recycling. The screen will make use of the fact that depletion of proteins needed for MPR recycling leads to dispersal of MPRs into peripherally localized cellular compartments. Computer software can detect this dispersal, permitting automated analysis of the effects of 22,000 siRNAs transfected into cultured cells. (3) Also proposed are experiments to further characterize two novel Rab9 effectors that are important for this trafficking pathway: RhoBTB3 and RUTBC1. In summary, these experiments open up entirely new areas of investigation in the area of MPR trafficking and will provide fundamental information regarding the mechanisms of receptor trafficking in human cells. The work has broad application to our understanding of a number of disease states including diabetes, cancer, heart disease and neurological disorders.

Public Health Relevance

Membrane traffic is essential for our ability to both secrete and respond to insulin, to clear cholesterol from the bloodstream, and for cells of the immune system to kill pathogens. Defects in membrane traffic underlie a number of disease states and virus infection depends upon this process. By understanding the molecular events responsible for membrane traffic, we will be better able to intervene in a variety of disease states.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK037332-28
Application #
8497668
Study Section
Membrane Biology and Protein Processing (MBPP)
Program Officer
Haft, Carol R
Project Start
1986-07-01
Project End
2014-06-30
Budget Start
2013-07-01
Budget End
2014-06-30
Support Year
28
Fiscal Year
2013
Total Cost
$416,747
Indirect Cost
$153,791
Name
Stanford University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
009214214
City
Stanford
State
CA
Country
United States
Zip Code
94305
Lu, Albert; Pfeffer, Suzanne R (2014) A CULLINary ride across the secretory pathway: more than just secretion. Trends Cell Biol 24:389-99
Lu, Albert; Pfeffer, Suzanne R (2013) Golgi-associated RhoBTB3 targets cyclin E for ubiquitylation and promotes cell cycle progression. J Cell Biol 203:233-50
Pfeffer, Suzanne R (2013) Rab GTPase regulation of membrane identity. Curr Opin Cell Biol 25:414-9
Brown, Frank C; Schindelhaim, Carmel H; Pfeffer, Suzanne R (2011) GCC185 plays independent roles in Golgi structure maintenance and AP-1-mediated vesicle tethering. J Cell Biol 194:779-87
Nottingham, Ryan M; Ganley, Ian G; Barr, Francis A et al. (2011) RUTBC1 protein, a Rab9A effector that activates GTP hydrolysis by Rab32 and Rab33B proteins. J Biol Chem 286:33213-22
Deffieu, Maika S; Pfeffer, Suzanne R (2011) Niemann-Pick type C 1 function requires lumenal domain residues that mediate cholesterol-dependent NPC2 binding. Proc Natl Acad Sci U S A 108:18932-6
Pfeffer, Suzanne R (2010) How the Golgi works: a cisternal progenitor model. Proc Natl Acad Sci U S A 107:19614-8
Pfeffer, Suzanne R (2010) Recent advances in understanding Golgi biogenesis. F1000 Biol Rep 2:32
Pfeffer, Suzanne R (2009) Multiple routes of protein transport from endosomes to the trans Golgi network. FEBS Lett 583:3811-6
Ganley, Ian G; Pfeffer, Suzanne R (2006) Cholesterol accumulation sequesters Rab9 and disrupts late endosome function in NPC1-deficient cells. J Biol Chem 281:17890-9

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