Research Interest

Membrane Traffic; Protein Degradation

A major aim of our work is to understand how cells regulate the delivery of proteins to their surface. In eukaryotic cells, secreted and cell surface proteins are transported from the site of synthesis in the endoplasmic reticulum (ER), through a series of intracellular compartments (ex: ERGIC and the Golgi) to the cell surface. The correct trafficking of digestive enzymes, neurotransmitters, hormones, morphogens, signal transducing molecules, adhesion proteins, etc is responsible for all human developmental and life processes. My laboratory is developing a “virtual” time and space map of all of the molecular events that regulate trafficking. We were the first to clone several proteins that are required for transport and are now using biochemical, morphological, molecular and genetic methods to define their exact functions. We concentrate on tethering factors that appear to link membranes prior to fusion and on a family of proteins with guanine nucleotide exchange activity that promote cargo selection during transport. Tethering proteins appear to act as molecular "Velcro" to facilitate correct membrane-membrane pairing. The exchange factors act as molecular "fly paper" that selects and keeps a patch of cargo for transport. The ultimate goal of our studies is to provide a detailed understanding of protein traffic at the molecular level as a basis for the development of disease-specific therapies that target the deficient steps in protein traffic. 

A complementary area of focus within our group is the control of protein degradation. Chaperones catalyze the correct folding of newly synthesized proteins in the ER. Incorrect or inefficient folding leads to the scavenging of the protein by the ER quality control system and its elimination by proteasomal degradation. We have developed an in vivo system, using the genetically tractable yeast, S. cerevisiae, to analyze the process. This allowed the identification of a multi-component sorting machinery in yeast that sequesters misfolded proteins in ER subdomains prior to their degradation. We are seeking to identify a similar system in mammalian cells. In addition, we are exploring the relationship between proteasomal degradation and a separate degradative pathway, autophagy. We have uncovered that overtaxing the proteasomal pathway by overloading the cell with misfolded proteins or by stress leads to upregulation of autophagic degradation. We are now investigating the molecular signaling that links the two pathways. the goal of this project is to develop target-based technology that will selectively slow the degradation of clinically relevant proteins.

A major focus in the lab that intersects the trafficking and degradative areas is the biogenesis of the Cystic Fibrosis Transconductance regulator (CFTR), a clinically important protein that is the causative agent of Cystic Fibrosis (CF). We are analyzing cellular proteins that regulate the triage decisions of either transporting the protein to the cell surface or degrading it. We have uncovered that components of the COPII coat are involved in targeting CFTR for degradation. In addition, we have used novel live-imaging approaches to reveal that CFTR degradation machinery is spatially restricted. CFTR is initially synthesized in the ER and misfolded proteins are initially removed from that compartment through proteasomal degradation. CFTR molecules that escape the ER-associated degradation (especially the ?F508 CFTR mutant that is responsible for the vast majority of CF cases) appear to accumulate in subdomains of the ER and are degraded through an autophagic pathway. We are now exploring molecular mechanisms of CFTR degradation in epithelial lung cells under conditions that mimic those that occur in the lungs of CF patients. Our findings have important impact on CF therapeutics since previous strategies focused exclusively on preventing proteasomal degradation. Our new insight suggests that therapies to rescue CF must also target autophagy.

Lab Members:
Karl Fu, Post-Doctoral Fellow
Robert Grabski, Graduate Student
Jason Lowry, Graduate Student
Melanie Styers, Post-Doctoral Fellow
Tomasz Szul, Graduate Student 
Cristy Towers, Graduate Student

Techniques Used:
Molecular: DNA cloning, PCR, random and point mutagenesis, production of recombinant proteins, in vitro transcription/translation, transformation, LacZ assays
Cellular: cell culture, immunofluorescence, transfection, cell-free assays, fluorescence activated cell sorting (FACS) analysis, real time green fluorescence protein imaging, antibody loading, immunoprecipitation, fluorescence recovery after photobleaching (FRAP), infection with viruses (HCMV, VSV, HSV)
Biochemical: ELISA, SDS-PAGE, Westerns, BIOCOR binding analysis, mass spectrometry, protein cross-linking
Genetic: yeast di-hybrid screens, analysis of mutant yeast strains, generation and analysis of transgenic Drosophila melanogaster

Available Rotation Projects:
Effect of aggresome formation on viral replication
Mapping interactive domains in the nucleotide exchange factor GBF and its activator p115
Mapping functional domains in the Ras-related exchange factor Rab1

Biography