9 research outputs found
Interaction of MDM33 with mitochondrial inner membrane homeostasis pathways in yeast
Membrane homeostasis affects mitochondrial dynamics, morphology, and function. Here we report genetic and proteomic data that reveal multiple interactions of Mdm33, a protein essential for normal mitochondrial structure, with components of phospholipid metabolism and mitochondrial inner membrane homeostasis. We screened for suppressors of MDM33 overexpression-induced growth arrest and isolated binding partners by immunoprecipitation of cross-linked cell extracts. These approaches revealed genetic and proteomic interactions of Mdm33 with prohibitins, Phb1 and Phb2, which are key components of mitochondrial inner membrane homeostasis. Lipid profiling by mass spectrometry of mitochondria isolated from Mdm33-overexpressing cells revealed that high levels of Mdm33 affect the levels of phosphatidylethanolamine and cardiolipin, the two key inner membrane phospholipids. Furthermore, we show that cells lacking Mdm33 show strongly decreased mitochondrial fission activity indicating that Mdm33 is critical for mitochondrial membrane dynamics. Our data suggest that MDM33 functionally interacts with components important for inner membrane homeostasis and thereby supports mitochondrial division
Bro1 binding to Snf7 regulates ESCRT-III membrane scission activity in yeast
The ubiquitin hydrolase activating factor Bro1 enhances ESCRT-III stability by inhibiting Vps4-mediated disassembly
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Regulation of ESCRT-III assembly and membrane scission activity in the budding yeast Saccharomyces cerevisiae
The sequential recruitment and assembly of endosomal sorting complexes required for transport (ESCRTs) at the endosomal membrane mediate the selection and clustering of cargoes into vesicles that bud into the lumen of the endosome. In addition to regulating this sorting process at endosomes, in mammalian cells ESCRTs are additionally required for the budding of many types of enveloped viruses, as well as the separation of cells during cytokinesis. These processes share a topologically similar membrane scission event facilitated by regulated ESCRT-III assembly at the cytoplasmic surface of the membrane to promote the formation and scission of internal vesicles. The Snf7 subunit of ESCRT-III in yeast binds directly to an auxiliary protein, Bro1. Like ESCRT-III, Bro1 is required for the formation of intralumenal vesicles at endosomes, but its role in membrane scission has remained uncharacterized. We show that overexpression of Bro1, or its N-terminal Bro1 domain that binds Snf7, enhances the stability of ESCRT-III. Bro1 binding to the Snf7 subunit of ESCRT-III additionally inhibits Vps4-mediated disassembly of ESCRT-III complexes in vivo and in vitro. This stabilization effect correlates with a reduced frequency in the budding of intralumenal vesicles within endosomes, and the appearance of vesicle budding profiles, vesicles that have not yet separated from the limiting endosomal membrane, as observed by electron microscopy and 3-D electron tomography. These results implicate Bro1 as a regulator of ESCRT-III assembly state, and additionally suggest that Vps4-mediated disassembly of the ESCRT-III complex is important for vesicle membrane scission. We also observed that deletion of ESCRT-III proteins Snf7 or Vps24, or overexpression of Bro1 or the Bro1 domain causes the accumulation of budded yeast cells by flow cytometry. While these proteins have been shown to participate in cell abscission in mammalian cells, a role for these proteins in cell division has not been established in yeast. Our findings suggest that ESCRT-III and Bro1 participate in the abscission step of cytokinesis in yeast, implicating them as conserved effectors of membrane scission
Applications of laser-polarized <sup>129</sup>Xe to biomolecular assays
The chemical shift sensitivity and significant signal enhancement afforded by laser-polarized 129Xe have motivated the application of 129Xe NMR to biological imaging and spectroscopy. Recent research done by our group has used laser-polarized 129Xe in biomolecular assays that detect ligand-binding events and distinguish protein conformations. The successful application of unfunctionalized and functionalized 129Xe NMR to in vitro biomolecular assays suggests the potential future use of a functionalized xenon biosensor for in vivo imaging
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Development of a Functionalized Xenon Biosensor
NMR-based biosensors that utilize laser-polarized xenon offer potential advantages beyond current sensing technologies. These advantages include the capacity to simultaneously detect multiple analytes, the applicability to in vivo spectroscopy and imaging, and the possibility of âremoteâ amplified detection. Here, we present a detailed NMR characterization of the binding of a biotin-derivatized caged-xenon sensor to avidin. Binding of âfunctionalizedâ xenon to avidin leads to a change in the chemical shift of the encapsulated xenon in addition to a broadening of the resonance, both of which serve as NMR markers of ligandâtarget interaction. A control experiment in which the biotin-binding site of avidin was blocked with native biotin showed no such spectral changes, confirming that only specific binding, rather than nonspecific contact, between avidin and functionalized xenon leads to the effects on the xenon NMR spectrum. The exchange rate of xenon (between solution and cage) and the xenon spinâlattice relaxation rate were not changed significantly upon binding. We describe two methods for enhancing the signal from functionalized xenon by exploiting the laser-polarized xenon magnetization reservoir. We also show that the xenon chemical shifts are distinct for xenon encapsulated in different diastereomeric cage molecules. This demonstrates the potential for tuning the encapsulated xenon chemical shift, which is a key requirement for being able to multiplex the biosensor