Honors Capstone Project
Date of Submission
Arts and Science
Capstone Prize Winner
Won Capstone Funding
Sciences and Engineering
Little is understood about the major functions subunit C of vacuolar proton-translocating ATPase (V-ATPase), a membrane-bound enzyme found in many eukaryotic cells that is responsible for pumping protons across its membrane in order to acidify either organelles or extracellular compartments. Subunit C is a known stator subunit and is required for assembly of the functional holoenzyme. In addition, it has been shown that subunit C interacts with actin of the cytoskeleton in the plasma membrane V-ATPases of Manduca sexta. However, the universality of this interaction—specifically its presence in organelle membrane V-ATPases—has yet to be determined. Consequently, the interaction between subunit C and actin was tested in Saccharomyces cerevisiae (baker’s yeast), a model eukaryotic organism in which properties of this potential interaction could be easily determined.
The interaction was initially detected by the genetic mating assay known as the two-hybrid method. In addition to detecting the interaction, the two-hybrid method was utilized in order to determine the precise subunit C binding site(s) on actin’s structure by using a set of mutants each having actin that contained a different point mutation in its monomeric structure. The mutated residues corresponding to the mutants that exhibited a disrupted subunit C-actin interaction were identified as participants in subunit C binding. These mutations all fell in two regions: either side of the actin monomer, indicating that subunit C binds to actin at these locations.
Co-immunoprecipitation confirmed the presence of an interaction between subunit C and actin. When subunit C was immunoprecipitated via a myc epitope tag, actin was detected in the immunoprecipitate upon being incubated with an anti-actin antibody. Little actin was present in the negative control, which did not contain subunit C, suggesting that actin was immunoprecipitated due to its interaction with subunit C.
These results not only have implications regarding V-ATPase activity but also for the dynamics of actin filaments. Because of actin’s prominence within a cell as part of the structural support system, it is known to interact with many cellular proteins to achieve multiple intracellular tasks. This particular interaction indicates that subunit C may be responsible for directing assembled V-ATPases to their appropriate organelle membranes or locations on the plasma membrane. Furthermore, results from this study revealed that subunit C most likely binds to either side of actin’s monomeric structure, which leaves actin-actin binding sites free and suggests that it is likely to bind to actin filaments. This is consistent with the findings of the M. sexta study, where subunit C was found to bind to both monomeric and filamentous actin and even promote actin polymerization. All in all, the observed interaction between subunit C of V-ATPase and actin could mean that subunit C not only regulates V-ATPase activity at the membrane through controlling its assembly, but also through controlling the number of V-ATPase complexes present at the membrane via actin binding. Moreover, these two approaches could very well be related, defining subunit C’s role as one of complex regulation of V-ATPase activity.
Blackley, Amanda, "Characterizing the Interaction between Subunit C of V-ATPase and Actin of the Cytoskeleton in Saccharomyces cerevisiae" (2008). Honors Capstone Projects - All. 501.
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