Regulated Assembly of the V-ATPase: Structural Insights, Physiological Function, and Control by Cellular Signaling Pathways.
vacuolar (H+)-ATPases (V-ATPases) are ATP-driven proton pumps composed of a peripheral
V1 domain and a membrane embedded V0 domain. Regulated assembly of V1 and V0 represents
an important regulatory mechanism for controlling V-ATPase activity in vivo. This thesis
investigates regulated assembly using both yeast as a model system and dendritic cells,
where V-ATPase activity is im... read moreportant for antigen processing. In yeast, regulated
assembly was investigated both structurally and by investigating cellular signaling
pathways which modulate assembly. Subunit a is a 100 kDa membrane protein that possesses
an N-terminal cytoplasmic domain and a C-terminal hydrophobic domain. The N-terminal
domain is critical for intracellular targeting and regulation of V-ATPase assembly.
Despite its importance, there is currently no high resolution structure for subunit a of
the V-ATPase. We have used homology modeling to construct a model of the N-terminal
domain of Vph1p, one of two isoforms of subunit a expressed in yeast. The results of
studies investigating accessibility of introduced cysteine residues to chemical
modification provide an experimental test of the proposed model and have identified
regions of the N-terminal domain of subunit a that likely serve as interfacial contact
sites with the peripheral V1 domain. Regulated assembly of the V-ATPase in yeast is
mediated by the Ras/cAMP/PKA pathway, and we tested the hypothesis that PKA-dependent
phosphorylation of the V-ATPase subunit C functions in this regulation. Site-directed
mutagenesis of consensus PKA phosphorylation sites in subunit C was performed. The
resulting mutant displayed normal assembly and glucose-dependent disassembly, indicating
that none of the PKA consensus sites on subunit C are required for glucose-dependent
regulation of V-ATPase assembly. Future plans to identify downstream mediators of PKA,
or the direct phosphorylation of other pump subunits, is discussed. Dendritic cells
function in antigen processing as part of the adaptive immune response. In dendritic
cells, cluster disruption induces a semi-mature phenotype associated with immune
tolerance. Thus, semi-mature dendritic cells are able to process and present
self-peptides to suppress autoimmune responses. We have investigated V-ATPase assembly
in bone marrow-derived, murine dendritic cells, and have observed an increase in
assembly and activity following cluster disruption. This increased assembly is not
dependent upon new protein synthesis and is regulated by a PI 3-kinase/mTOR dependent
pathway. The significance of this finding for the treatment of autoimmune diseases is
Thesis (Ph.D.)--Tufts University, 2014.
Submitted to the Dept. of Cellular & Molecular Physiology.
Advisor: Michael Forgac.
Committee: Laura Liscum, Peter Juo, Alexander Poltorak, Brent Cochran, and Sylvie Breton.
Keywords: Physiology, and Biochemistry.read less