Endocytic Internalization in Budding Yeast Requires Coordinated Actin Nucleation and Myosin Motor Activity
ABSTRACT Actin polymerization essential for endocytic internalization in budding yeast is controlled by four nucleation promoting factors (NPFs) that each exhibits a unique dynamic behavior at endocytic sites. How each NPF functions and is regulated to restrict actin assembly to late stages of endocytic internalization is not known. Quantitative analysis of NPF biochemical activities, and genetic analysis of recruitment and regulatory mechanisms, defined a linear pathway in which protein composition changes at endocytic sites control actin assembly and function. We show that yeast WASP initiates actin assembly at endocytic sites and that this assembly and the recruitment of a yeast WIP-like protein by WASP recruit a type I myosin with both NPF and motor activities. Importantly, type I myosin motor and NPF activities are separable, and both contribute to endocytic coat inward movement, which likely represents membrane invagination. These results reveal a mechanism in which actin nucleation and myosin motor activity cooperate to promote endocytic internalization.
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ABSTRACT: To determine the mechanism of resistance to the fungicide phenamacril (JS399-19) in Fusarium graminearum, the causal agent of Fusarium head blight, we sequenced and annotated the genome of the resistant strain YP-1 (generated by treating the F. graminearum reference strain PH-1 with phenamacril). Of 1.4 million total reads from an Illumina-based paired-end sequencing assay, 92.80% were aligned to the F. graminearum reference genome. Compared with strain PH-1, strain YP-1 contained 1,989 single-nucleotide polymorphisms that led to amino acid mutations in 132 genes. We sequenced 22 functional annotated genes of another F. graminearum sensitive strain (strain 2021) and corresponding resistant strains. The only mutation common to all of the resistant mutants occurred in the gene encoding myosin-5 (point mutations at codon 216, 217, 418, 420, or 786). To confirm whether the mutations in myosin-5 confer resistance to phenamacril, we exchanged the myosin-5 locus between the sensitive strain 2021 and the resistant strain Y2021A by homologous double exchange. The transformed mutants with a copy of the resistant fragment exhibited resistance to phenamacril, and the transformed mutant with a copy of the sensitive fragment exhibited sensitivity to phenamacril. These results indicate that mutations in myosin-5 confers resistance to phenamacril in F. graminearum.Scientific Reports 02/2015; 5:8248. DOI:10.1038/srep08248 · 5.08 Impact Factor
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ABSTRACT: eLife digest Cells take up proteins and other useful material (called cargo) from their external environment through a process known as endocytosis. To start with, the cargo accumulates in a patch on the surface of the cell. On the inner side of the cell's membrane, a protein called clathrin gathers around the patch of cargo. Clathrin molecules and many other proteins bind together to make a lattice-like coat that causes the membrane to curve inwards and form a pocket that contains the cargo. This continues until the cargo is completely surrounded by membrane and eventually forms a bubble-like structure, or ‘vesicle’, that moves into the cell. More than 50 other proteins are involved in the endocytosis. These proteins arrive at the site of endocytosis in a particular order, complete their tasks and then move away to be used in further rounds of endocytosis. It is not clear how these proteins are organized to complete these steps because it is technically difficult to track the movements of many proteins at the same time. Here, Picco et al. developed a new fluorescence microscopy method that enabled them to track the positions of many of the proteins involved in endocytosis in yeast cells in real time. The experiments revealed when the proteins arrived at the site of endocytosis and how they assembled in relation to the membrane. For example, a group of proteins called N-BAR proteins formed an extended lattice covering the sides of the pocket that forms as the membrane curves inwards. To transform the flat membrane into a vesicle, a network of filaments made of a protein called actin needs to form at the site of endocytosis. The new method shows that the actin filaments grow in a small region at the base of the developing vesicle. By combining different types of microscopy data, Picco et al. were able to build a comprehensive model describing when the proteins involved in endocytosis move and assemble. The next challenge will be to understand the physics behind the molecular machine composed of these many proteins and the cell membrane. DOI: http://dx.doi.org/10.7554/eLife.04535.002eLife Sciences 02/2015; 4. DOI:10.7554/eLife.04535 · 8.52 Impact Factor
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ABSTRACT: Endocytosis, the process whereby the plasma membrane invaginates to form vesicles, is essential for bringing many substances into the cell and for membrane turnover. The mechanism driving clathrin-mediated endocytosis (CME) involves > 50 different protein components assembling at a single location on the plasma membrane in a temporally ordered and hierarchal pathway. These proteins perform precisely choreographed steps that promote receptor recognition and clustering, membrane remodeling, and force-generating actin-filament assembly and turnover to drive membrane invagination and vesicle scission. Many critical aspects of the CME mechanism are conserved from yeast to mammals and were first elucidated in yeast, demonstrating that it is a powerful system for studying endocytosis. In this review, we describe our current mechanistic understanding of each step in the process of yeast CME, and the essential roles played by actin polymerization at these sites, while providing a historical perspective of how the landscape has changed since the preceding version of the YeastBook was published 17 years ago (1997). Finally, we discuss the key unresolved issues and where future studies might be headed. Copyright © 2015 by the Genetics Society of America.Genetics 02/2015; 199(2):315-358. DOI:10.1534/genetics.112.145540 · 4.87 Impact Factor