The full text of this item is not available at this time because the student has placed this item under an embargo for a period of time. The Libraries are not authorized to provide a copy of this work during the embargo period, even for Texas A&M users with NetID.
Mechanism of Membrane Fission by Yeast Amphiphysin
Abstract
Solutes and nutrients are internalized by endocytosis and delivered to early endosomes in membranous transport carriers in eukaryotic cells. The release of these carriers into the cell requires membrane fission proteins. One well-characterized membrane fission protein is dynamin-1, a protein that releases clathrin-coated endocytic vesicles in neurons. Dynamin-1 induces membrane fission through conformation changes of a helical polymer upon GTP hydrolysis. In contrast, endocytosis in S. cerevisiae requires an N-BAR homolog, Rvs161/167p. While targeted actin polymerization pulls the endocytic bud to fission, Rvs161/167p assemble on the highly curved membrane tubule just before the release of the endocytic vesicle.
At the same time, N-BAR proteins, like amiphiphysin and endophilin, can induce membrane fission in vitro [1, 2]. However, the biological role of this fission activity is unclear. C. elegans N-BAR protein, AMPH-1 performs an unexpected GTP-stimulated membrane fission activity. Here, using a single-particle fluorescent burst assay, we report a novel GTP-stimulated fission mechanism of C. elegans N-BAR protein, AMPH-1. The focus of this research is the characterization of Rvs161/167p as a potent membrane fission agent. For this work, we have employed a single particle fluorescent burst assay to quantitatively measure the fission activity of yeast N-BAR. Using functional mutants [3], we show the binding activity of the Rvs161/167p BAR domains can be separated. Charge-reversal mutations of two conservative lys on the concave surface of the BAR domains result in inefficient membrane binding, with limited tubulation and fission activity. In contrast, either a deletion or disturbance in the amphipathicity of the N-terminal helices results in a complete loss of membrane tubulation and fission activity, despite the residual binding activity. This work thus systematically analyzes membrane fission by an N-BAR protein from binding, to tubulation, and to fission. This study confirms that Rvs161/167p causes membrane fission using its amphipathic helix, which is likely inserted into the membrane to create membrane curvature. Although the Rvs161/167p BAR domains were found to be important for binding, both membrane binding and tubulation were more strongly impacted by the N-terminal, amphipathic α-helices.
Citation
Gong, Xue (2022). Mechanism of Membrane Fission by Yeast Amphiphysin. Doctoral dissertation, Texas A&M University. Available electronically from https : / /hdl .handle .net /1969 .1 /198085.