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      Overlap of cargo binding sites on myosin V coordinates the inheritance of diverse cargoes

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          Abstract

          Vacuole- and mitochondria-specific cargo adaptors compete for an overlapping binding site on Myo2 to determine the inheritance of these organelles during budding.

          Abstract

          During cell division, organelles are distributed to distinct locations at specific times. For the yeast vacuole, the myosin V motor, Myo2, and its vacuole-specific cargo adaptor, Vac17, regulate where the vacuole is deposited and the timing of vacuole movement. In this paper, we show that Mmr1 functions as a mitochondria-specific cargo adaptor early in the cell cycle and that Mmr1 binds Myo2 at the site that binds Vac17. We demonstrate that Vac17 and Mmr1 compete for binding at this site. Unexpectedly, this competition regulates the volume of vacuoles and mitochondria inherited by the daughter cell. Furthermore, eight of the nine known Myo2 cargo adaptors overlap at one of two sites. Vac17 and Mmr1 overlap at one site, whereas Ypt11 and Kar9 bind subsets of residues that also bind Ypt31/Ypt32, Sec4, and Inp2. These observations predict that competition for access to Myo2 may be a common mechanism to coordinate the inheritance of diverse cargoes.

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          Genomic libraries and a host strain designed for highly efficient two-hybrid selection in yeast.

          The two-hybrid system is a powerful technique for detecting protein-protein interactions that utilizes the well-developed molecular genetics of the yeast Saccharomyces cerevisiae. However, the full potential of this technique has not been realized due to limitations imposed by the components available for use in the system. These limitations include unwieldy plasmid vectors, incomplete or poorly designed two-hybrid libraries, and host strains that result in the selection of large numbers of false positives. We have used a novel multienzyme approach to generate a set of highly representative genomic libraries from S. cerevisiae. In addition, a unique host strain was created that contains three easily assayed reporter genes, each under the control of a different inducible promoter. This host strain is extremely sensitive to weak interactions and eliminates nearly all false positives using simple plate assays. Improved vectors were also constructed that simplify the construction of the gene fusions necessary for the two-hybrid system. Our analysis indicates that the libraries and host strain provide significant improvements in both the number of interacting clones identified and the efficiency of two-hybrid selections.
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            An ER-mitochondria tethering complex revealed by a synthetic biology screen.

            Communication between organelles is an important feature of all eukaryotic cells. To uncover components involved in mitochondria/endoplasmic reticulum (ER) junctions, we screened for mutants that could be complemented by a synthetic protein designed to artificially tether the two organelles. We identified the Mmm1/Mdm10/Mdm12/Mdm34 complex as a molecular tether between ER and mitochondria. The tethering complex was composed of proteins resident of both ER and mitochondria. With the use of genome-wide mapping of genetic interactions, we showed that the components of the tethering complex were functionally connected to phospholipid biosynthesis and calcium-signaling genes. In mutant cells, phospholipid biosynthesis was impaired. The tethering complex localized to discrete foci, suggesting that discrete sites of close apposition between ER and mitochondria facilitate interorganelle calcium and phospholipid exchange.
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              Accuracy and precision in quantitative fluorescence microscopy

              The light microscope has long been used to document the localization of fluorescent molecules in cell biology research. With advances in digital cameras and the discovery and development of genetically encoded fluorophores, there has been a huge increase in the use of fluorescence microscopy to quantify spatial and temporal measurements of fluorescent molecules in biological specimens. Whether simply comparing the relative intensities of two fluorescent specimens, or using advanced techniques like Förster resonance energy transfer (FRET) or fluorescence recovery after photobleaching (FRAP), quantitation of fluorescence requires a thorough understanding of the limitations of and proper use of the different components of the imaging system. Here, I focus on the parameters of digital image acquisition that affect the accuracy and precision of quantitative fluorescence microscopy measurements.
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                Author and article information

                Journal
                J Cell Biol
                J. Cell Biol
                jcb
                The Journal of Cell Biology
                The Rockefeller University Press
                0021-9525
                1540-8140
                9 July 2012
                : 198
                : 1
                : 69-85
                Affiliations
                [1 ]Program in Cell and Molecular Biology , [2 ]Life Sciences Institute , and [3 ]Department of Cell and Developmental Biology, University of Michigan Medical School, Ann Arbor, MI 48109
                Author notes
                Correspondence to Lois S. Weisman: lweisman@ 123456umich.edu
                Article
                201201024
                10.1083/jcb.201201024
                3392941
                22753895
                c151ffab-dad9-40ad-bd02-b443fc59c4b5
                © 2012 Eves et al.

                This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/3.0/).

                History
                : 6 January 2012
                : 31 May 2012
                Categories
                Research Articles
                Article

                Cell biology
                Cell biology

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