The movement of protein "cargo" in vesicles between successive intracellular compartments requires "zip code" information to link a vesicle with the appropriate acceptor membrane. This recognition appears to be provided by tethering factors, which can generally be divided into two groups: long coiled-coil proteins and large multi-subunit complexes. Tethers may act as molecular nets to catch relevant vesicles and increase the possibility that they will fuse with the appropriate membrane. The necessity for tethering factors in accurate trafficking of vesicles appears conserved from yeast to man, and tethering factors have been identified for most membrane traffic steps. The long-term goal of this laboratory is to understand the interplay between tethering factors, Rab proteins and SNARE proteins in tethering, docking, and fusion of transport intermediates and to determine their mechanisms of action in different stages of protein trafficking. This project aims to investigate the precise functions of the conserved oligomeric Golgi (COG) protein complex in budding yeast. The work operates under the main hypothesis that the COG complex interacts with a sub-set of Golgi Rab and SNARE proteins to work as a tether that effects high-fidelity docking and fusion of vesicles moving cargo between Golgi compartments. There are three aims to this project. Aim 1. Many state-of-the-art proteomic strategies and technologies have been used to identify a set of COG-interacting proteins (COG "interactome"). It is necessary now to sort out this knowledge and determine which interacting proteins are essential for COG complex functions and how different COG complex subunits communicate with their protein partners. Yeast genetic methods combined with biochemical and cell biological approaches will be used to address these questions. Initial studies will be focused on the analysis of COG-SNARE and COG-Rab interactions. Aim 2. All COG complex subunits are peripheral-membrane proteins and most likely accomplish their function through interaction with both transmembrane and peripheral-membrane proteins. Therefore, an important question is what molecular interactions determine membrane association of the COG complex. An in vitro system will be employed to identify all molecules, including membrane lipids, responsible for COG-membrane interaction. Aim 3 is to reconstitute the entire COG-dependent steps of membrane trafficking in vitro. This is an ambitious set of experiments that would directly test the functions of both the COG complex and COG complex-interacting proteins and would aid in deciphering the molecular mechanisms of COG complex action. Together these aims constitute a detailed structural-functional analysis of the machinery that determines high-fidelity bidirectional protein and lipid trafficking in the Golgi apparatus. The advanced in vivo and in vitro approaches to defining the precise mechanisms of the Golgi vesicle tethering functions will be applicable to related processes in the wider field of membrane trafficking. This project will integrate research with education and enrichment of cultural diversity. The PI teaches in several graduate courses, including a new interdisciplinary graduate cell biology course that he developed. In addition, he interacts with faculty and students at nearby undergraduate institutions such as the University of Arkansas Little Rock, University of Central Arkansas, Hendrix University and the University of Arkansas Community College at Batesville. A Biosciences Research Infrastructure Network (BRIN) project promotes a formal summer undergraduate program at UAMS. Students from institutions such as Hendrix University and Central High School in Little Rock, which include students from groups traditionally underrepresented in science, will participate in this project.
Eukaryotic cells are compartmentalized - consist of several specialized functional subunits, called organelles – that make them distinct from prokaryotic cells. Golgi apparatus is the specific organelle that plays an important role in protein sorting and modifications. Vladimir Lupashin’s laboratory has been involved for several years in the understanding of the mechanisms of protein trafficking between Golgi sub-compartments. Protein transport through the secretory pathway occurs via transport vesicles under the direction of a large set of protein components. The movement of "cargo" between successive intracellular compartments requires "zip code" information that links a vesicle with the appropriate acceptor membrane. This initial recognition is provided by vesicle tethering factors, which provide a molecular net to catch relevant vesicles and increase the possibility that they will fuse with the appropriate membrane. The long-term goal of this project is to understand the interplay between tethering factors and other components of vesicle fusion machinery and to elucidate their mechanisms of action in different stages of membrane trafficking. The COG complex is a Golgi located vesicle tethering factor that consist of eight subunits named COG1 through COG8. It is important to understand the COG complex function because COG is a key Golgi trafficking regulator. Mutations in COG subunits observed in yeasts, flies, worms, plants and human lead to severe defects on both cellular and organismal levels. At the molecular level, the Lupashin’s laboratory discovered that the COG complex physically and functionally interacts with five Golgi SNARE molecules – core components of the Golgi membrane fusion machinery. COG complex is responsible for intracellular localization and activity of these fusogenic molecules. Another set of interactions was discovered between the COG complex and resident proteins of transport vesicles. These discoveries suggest the mechanism by which the COG complex is orchestrating capturing of relevant membrane carriers and their fusion with the exact Golgi sub-compartment. On a cellular level, the role of the COG complex has been investigated using a novel intra-Golgi trafficking assay that biochemically and microscopically measure plasma membrane-to –ER delivery of bacterial subtilase cytotoxin. COG-dependent stability and localization of major Golgi-localized protein modifying enzymes was elucidated. Finally, a structural basis for a human glycosylation disorder caused by mutation of the COG4 gene was revealed. This project also provided an excellent experience in science to high school students, graduate students and professional researchers. Four graduate student, three undergraduate student, four high school students and a visiting educational college faculty were intimately involved in this project. They have learned a vast variety of microbiological, molecular, biochemical and microscopic techniques, have gotten experience in obtaining and critically analyzing data as well as preparing results for both oral and poster presentation. Students wrote several scientific papers and presented 4 oral and 12 poster presentations at both national and international meetings
