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. We have shown that the protein, GCC185 is needed for tethering of MPRs at the Golgi. To investigate the mechanism of MPR vesicle tethering at the Golgi, we will analyze the structure of this purified Golgi tether using atomic force microscopy. This is important because transport vesicle tethering is a fundamental cell biological process that is poorly understood. We will test if the protein bends on the Golgi, in cells, and whether this bending is needed for its function. We will also determine the consequence of Rab GTPase binding on GCC185's conformation. This is important because GTPase binding is a common feature of Golgins and is likely to reflect the most physiological state of these proteins. We will next add fluorescently labeled, mannose 6-phosphate receptor-containing vesicles and monitor how these engage the GCC185 tether. Where do the vesicles bind? What models best explain how vesicles are tethered at the Golgi? Finally, we will study the very first step in retrograde transport: the loading of Rab9 onto late endosomes. For this, we will determine if Rab9A-specific DENND2 GEFs are part of a late endosomal Rab cascade. Understanding how DENND2 proteins first activate Rab9 will provide key information regarding establishment of the MPR retrograde trafficking pathway on late endosomes and formation of the late endocytic pathway. 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
High Priority, Short Term Project Award (R56)
Project #
2R56DK037332-29
Application #
8845329
Study Section
Membrane Biology and Protein Processing (MBPP)
Program Officer
Haft, Carol R
Project Start
1986-07-01
Project End
2015-06-30
Budget Start
2014-08-21
Budget End
2015-06-30
Support Year
29
Fiscal Year
2014
Total Cost
$120,375
Indirect Cost
$45,375
Name
Stanford University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
009214214
City
Stanford
State
CA
Country
United States
Zip Code
94305
Li, Jian; Pfeffer, Suzanne R (2016) Lysosomal membrane glycoproteins bind cholesterol and contribute to lysosomal cholesterol export. Elife 5:
Pfeffer, Suzanne R (2016) Lipoprotein secretion: It takes two to TANGO. J Cell Biol 213:297-9
Johnson, Tory A; Pfeffer, Suzanne R (2016) Ezetimibe-sensitive cholesterol uptake by NPC1L1 protein does not require endocytosis. Mol Biol Cell 27:1845-52
Cheung, Pak-yan Patricia; Limouse, Charles; Mabuchi, Hideo et al. (2015) Protein flexibility is required for vesicle tethering at the Golgi. Elife 4:
Li, Jian; Deffieu, Maika S; Lee, Peter L et al. (2015) Glycosylation inhibition reduces cholesterol accumulation in NPC1 protein-deficient cells. Proc Natl Acad Sci U S A 112:14876-81
Cheung, Pak-Yan Patricia; Pfeffer, Suzanne R (2015) Molecular and cellular characterization of GCC185: a tethering protein of the trans-Golgi network. Methods Mol Biol 1270:179-90
Lee, Peter L; Ohlson, Maikke B; Pfeffer, Suzanne R (2015) Rab6 regulation of the kinesin family KIF1C motor domain contributes to Golgi tethering. Elife 4:
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 (2012) Rab GTPase localization and Rab cascades in Golgi transport. Biochem Soc Trans 40:1373-7
van Rahden, Vanessa A; Brand, Kristina; Najm, Juliane et al. (2012) The 5-phosphatase OCRL mediates retrograde transport of the mannose 6-phosphate receptor by regulating a Rac1-cofilin signalling module. Hum Mol Genet 21:5019-38

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