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Sancak, Yasemin

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Sancak

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Yasemin

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Sancak, Yasemin
Author's Publications: search.filters.isAuthorOfPublication.ec70e051-68df-416e-924c-344d4e8d5511

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Now showing 1 - 4 of 4
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    Engineered ascorbate peroxidase as a genetically-encoded reporter for electron microscopy
    (2013) Martell, Jeffrey D.; Deerinck, Thomas J.; Sancak, Yasemin; Poulos, Thomas L.; Mootha, Vamsi; Sosinsky, Gina E.; Ellisman, Mark H.; Ting, Alice Y.
    Electron microscopy (EM) is the standard method for imaging cellular structures with nanometer resolution, but existing genetic tags are inactive in most cellular compartments1 or require light and are difficult to use2. Here we report the development of a simple and robust EM genetic tag, called “APEX,” that is active in all cellular compartments and does not require light. APEX is a monomeric 28 kDa peroxidase that withstands strong EM fixation to give excellent ultrastructural preservation. We demonstrate the utility of APEX for high-resolution EM imaging of a variety of mammalian organelles and specific proteins. We also fused APEX to the N- or C-terminus of the mitochondrial calcium uniporter (MCU), a newly identified channel whose topology is disputed3,4. MCU-APEX and APEX-MCU give EM contrast exclusively in the mitochondrial matrix, suggesting that both the N-and C-termini of MCU face the matrix.
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    MCU encodes the pore conducting mitochondrial calcium currents
    (eLife Sciences Publications, Ltd, 2013) Chaudhuri, Dipayan; Sancak, Yasemin; Mootha, Vamsi; Clapham, David
    Mitochondrial calcium (Ca2+) import is a well-described phenomenon regulating cell survival and ATP production. Of multiple pathways allowing such entry, the mitochondrial Ca2+ uniporter is a highly Ca2+-selective channel complex encoded by several recently-discovered genes. However, the identity of the pore-forming subunit remains to be established, since knockdown of all the candidate uniporter genes inhibit Ca2+ uptake in imaging assays, and reconstitution experiments have been equivocal. To definitively identify the channel, we use whole-mitoplast voltage-clamping, the technique that originally established the uniporter as a Ca2+ channel. We show that RNAi-mediated knockdown of the mitochondrial calcium uniporter (MCU) gene reduces mitochondrial Ca2+ current (IMiCa), whereas overexpression increases it. Additionally, a classic feature of IMiCa, its sensitivity to ruthenium red inhibition, can be abolished by a point mutation in the putative pore domain without altering current magnitude. These analyses establish that MCU encodes the pore-forming subunit of the uniporter channel. DOI: http://dx.doi.org/10.7554/eLife.00704.001
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    Architecture of the Mitochondrial Calcium Uniporter
    (2016) Oxenoid, Kirill; Dong, Ying; Cao, Chan; Cui, Tanxing; Sancak, Yasemin; Markhard, Andrew L.; Grabarek, Zenon; Kong, Liangliang; Liu, Zhijun; Ouyang, Bo; Cong, Yao; Mootha, Vamsi; Chou, James
    Mitochondria from multiple, eukaryotic clades uptake and buffer large amounts of calcium (Ca2+) via an inner membrane transporter called the uniporter. Early studies demonstrated that this transport requires a mitochondrial membrane potential and that the uniporter is itself Ca2+ activated, and blocked by ruthenium red or Ru3601. Later, electrophysiological studies demonstrated that the uniporter is an ion channel with remarkably high conductance and selectivity2. Ca2+ entry into mitochondria is also known to activate the TCA cycle and appears to be critical for matching ATP production in mitochondria with its cytosolic demand3. MCU (mitochondrial calcium uniporter) is the pore forming and Ca2+ conducting subunit of the uniporter, but its primary sequence does not resemble any calcium channel known to date. Here, we report the structure of the core region of MCU, determined using nuclear magnetic resonance (NMR) and electron microscopy (EM). MCU is a homo-oligomer with the second transmembrane helix forming a hydrophilic pore across the membrane. The channel assembly represents a new solution of ion channel architecture and is stabilized by a coiled coil motif protruding in the mitochondrial matrix. The critical DxxE motif forms the pore entrance featuring two carboxylate rings, which appear to be the selectivity filter based on the ring dimensions and functional mutagenesis. To our knowledge, this is one of the largest structures characterized by NMR, which provides a structural blueprint for understanding the function of this channel.
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    MICU2, a Paralog of MICU1, Resides within the Mitochondrial Uniporter Complex to Regulate Calcium Handling
    (Public Library of Science, 2013) Plovanich, Molly; Bogorad, Roman L.; Sancak, Yasemin; Kamer, Kimberli; Strittmatter, Laura Anne; Li, Andrew A.; Girgis, Hany S.; Kuchimanchi, Satya; De Groot, Jack; Speciner, Lauren; Taneja, Nathan; OShea, Jonathan; Koteliansky, Victor; Mootha, Vamsi
    Mitochondrial calcium uptake is present in nearly all vertebrate tissues and is believed to be critical in shaping calcium signaling, regulating ATP synthesis and controlling cell death. Calcium uptake occurs through a channel called the uniporter that resides in the inner mitochondrial membrane. Recently, we used comparative genomics to identify MICU1 and MCU as the key regulatory and putative pore-forming subunits of this channel, respectively. Using bioinformatics, we now report that the human genome encodes two additional paralogs of MICU1, which we call MICU2 and MICU3, each of which likely arose by gene duplication and exhibits distinct patterns of organ expression. We demonstrate that MICU1 and MICU2 are expressed in HeLa and HEK293T cells, and provide multiple lines of biochemical evidence that MCU, MICU1 and MICU2 reside within a complex and cross-stabilize each other's protein expression in a cell-type dependent manner. Using in vivo RNAi technology to silence MICU1, MICU2 or both proteins in mouse liver, we observe an additive impairment in calcium handling without adversely impacting mitochondrial respiration or membrane potential. The results identify MICU2 as a new component of the uniporter complex that may contribute to the tissue-specific regulation of this channel.