Golgi associated Rab proteins - small GTPases of the Ras superfamily -- regulate critical, yet poorly understood, aspects of Golgi biogenesis, maintenance, and inheritance through affecting vesicle trafficking. The general (i.e., """"""""textbook"""""""") paradigm holds that Golgi Rab proteins regulate vesicle targeting via the recruitment and activation of tethering factors that mediate initial steps of membrane fusion. Our Preliminary Studies support a different and novel mechanism. We screened for molecular genetic relationship(s) between Rab33b and Rab6 - two Golgi Rabs that our works implicates in an intra-Golgi Rab cascade -- and the putative tether proteins, COG and ZW10/RINT-1, central to retrograde Golgi trafficking. We found that both Rab6 and Rab33b acted upstream of, not through, the tether complexes. By electron microscope tomography (ET), we observed that Rab6 knockdown results in an apparent inhibition of Golgi vesicle budding/transport as evidenced by the pronounced accumulation of both coated and uncoated budding profiles/vesicles along Golgi cisternal membranes. This has led us to conclude that Rab6 is required for efficient Golgi vesicle release/scission. In sum, our results suggest that Golgi Rabs such as Rab6 and Rab33b serve important functions in very early events at the Golgi related to vesicle scission, and that they act upstream of tether factor recruitment in this fundamental process. Based on our Preliminary Studies, we thus contend that a critical and functionally important role of Golgi-associated Rabs (such as Rab6 and Rab33b) is to regulate, through context sensitive effector sets, the budding/release of distinct classes of transport vesicles from Golgi cisternae. Functionally, we predict that each vesicle class supports a distinct trafficking pathway(s) and as such are compositionally distinct. Cellularly, we hypothesize that cisternal Rab proteins such as Rab6 and likely Rab33b play an important/central role in Golgi homeostasis. This hypothesis is supported by our finding that compared to control cells, Rab6 depleted cells reproducibly reveal a significant increase in the number of Golgi cisternae per stack (4.2 + 0.32 versus 6.8 + 0.46, respectively). We propose that Rab proteins regulate the distribution of Golgi resident proteins between cisternae. We predict that in Rab knockdown experiments that the distribution of individual Golgi enzymes will be shifted cis- or trans-ward. Mechanistically, we hypothesize that distinct effectors, including already identified candidate protein and others novel, will modulate the budding of individual vesicle classes. We propose the following three Specific Aims to test these hypotheses.
Specific Aim 1. We will test the hypothesis that Golgi associated Rab proteins such as Rab6 and Rab33b regulate the formation and/or release of multiple, distinct classes of vesicles from Golgi cisternae.
Specific Aim 2. We will test the hypothesis that Golgi-associated Rabs regulate the distribution of Golgi resident proteins between cisternae.
Specific Aim 3. We will test the hypothesis that mechanistically distinct protein sets, i.e., effectors, modulate the budding/release of individual vesicle classes. Characterization of structural/functional relationships within the mammalian Golgi apparatus and its regulatory pathways is important to human health. We and other investigators have found that such pathways are useful portals into the cell for the delivery of cell-killing reagents and antigens. Defects in these pathways can lead to human disease. Moreover, important machinery components in these pathways are involved in such processes as virus infection and aging. Modulation of Golgi associated Rabs may prove to be an important therapeutic approach.

Public Health Relevance

The Golgi apparatus is the central organelle within the secretory pathway of human cells. How the structure and function of this organelle is regulated is central to the health of cells and hence to the health humans. We find that Rab proteins, small GTPases of the Ras superfamily, regulate Golgi organization, likely at the level of vesicle formation/release/transport, a surprising outcome. Defects in Golgi associated Rab protein function have been already shown to affect vision (glaucoma), neurodegenerative disease, Chlamydial infection, viral entry and aging. Retrograde trafficking through the Golgi apparatus can be used as a portal for drug and antigen delivery. Rab modulation by small molecules may be a novel approach to controlling infection and other disease problems. Understanding the role of Rab proteins in structure and function relationships within the human Golgi apparatus should indeed further our understanding of human health.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM092960-04
Application #
8723844
Study Section
Membrane Biology and Protein Processing Study Section (MBPP)
Program Officer
Ainsztein, Alexandra M
Project Start
2011-09-30
Project End
2015-08-31
Budget Start
2014-09-01
Budget End
2015-08-31
Support Year
4
Fiscal Year
2014
Total Cost
Indirect Cost
Name
University of Arkansas for Medical Sciences
Department
Physiology
Type
Schools of Medicine
DUNS #
City
Little Rock
State
AR
Country
United States
Zip Code
72205
Goud, Bruno; Liu, Shijie; Storrie, Brian (2018) Rab proteins as major determinants of the Golgi complex structure. Small GTPases 9:66-75
Krokowski, Dawid; Guan, Bo-Jhih; Wu, Jing et al. (2017) GADD34 Function in Protein Trafficking Promotes Adaptation to Hyperosmotic Stress in Human Corneal Cells. Cell Rep 21:2895-2910
Yadav, Shilpi; Williamson, Jonathan K; Aronova, Maria A et al. (2017) Golgi proteins in circulating human platelets are distributed across non-stacked, scattered structures. Platelets 28:400-408
Siegel, Nisan; Lupashin, Vladimir; Storrie, Brian et al. (2016) High-magnification super-resolution FINCH microscopy using birefringent crystal lens interferometers. Nat Photonics 10:802-808
MacDonald, Laura; Baldini, Giulia; Storrie, Brian (2015) Does super-resolution fluorescence microscopy obsolete previous microscopic approaches to protein co-localization? Methods Mol Biol 1270:255-75
Liu, Shijie; Storrie, Brian (2015) How Rab proteins determine Golgi structure. Int Rev Cell Mol Biol 315:1-22
Majeed, Waqar; Liu, Shijie; Storrie, Brian (2014) Distinct sets of Rab6 effectors contribute to ZW10--and COG-dependent Golgi homeostasis. Traffic 15:630-47
Liu, Shijie; Hunt, Lauren; Storrie, Brian (2013) Rab41 is a novel regulator of Golgi apparatus organization that is needed for ER-to-Golgi trafficking and cell growth. PLoS One 8:e71886
Micaroni, Massimo; Stanley, Amanda C; Khromykh, Tatiana et al. (2013) Rab6a/a' are important Golgi regulators of pro-inflammatory TNF secretion in macrophages. PLoS One 8:e57034
Liu, Shijie; Storrie, Brian (2012) Are Rab proteins the link between Golgi organization and membrane trafficking? Cell Mol Life Sci 69:4093-106

Showing the most recent 10 out of 12 publications