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Denic, Vladimir

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Denic

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Vladimir

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Denic, Vladimir

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2009 - 200912010 - 20175

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Author's Publications: search.filters.isAuthorOfPublication.72366d05-4e0a-473c-b155-bd62413b1f4e

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Now showing 1 - 6 of 6
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    Defining the Essential Function of Yeast Hsf1 Reveals a Compact Transcriptional Program for Maintaining Eukaryotic Proteostasis
    (Elsevier BV, 2016) Solis, Eric; Pandey, Jai P.; Zheng, Xu; Jin, Dexter X.; Gupta, Piyush B.; Airoldi, Edoardo; Pincus, David Frank; Denic, Vladimir
    Despite its eponymous association with the heat shock response, yeast heat shock factor 1 (Hsf1) is essential even at low temperatures. Here we show that engineered nuclear export of Hsf1 results in cytotoxicity associated with massive protein aggregation. Genome-wide analysis revealed that Hsf1 nuclear export immediately decreased basal transcription and mRNA expression of 18 genes, which predominately encode chaperones. Strikingly, rescuing basal expression of Hsp70 and Hsp90 chaperones enabled robust cell growth in the complete absence of Hsf1. With the exception of chaperone gene induction, the vast majority of the heat shock response was Hsf1-independent. By comparative analysis of mammalian cell lines, we found that only heat shock-induced but not basal expression of chaperones is dependent on the mammalian Hsf1 homolog (HSF1). Our work reveals that yeast chaperone gene expression is an essential housekeeping mechanism and provides a roadmap for defining the function of HSF1 as a driver of oncogenesis.
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    Receptor-Bound Targets of Selective Autophagy Use a Scaffold Protein to Activate the Atg1 Kinase
    (Elsevier BV, 2015) Kamber, Roarke Alexander; Shoemaker, Christopher; Denic, Vladimir
    Selective autophagy eliminates protein aggregates, damaged organelles, and other targets that otherwise accumulate and cause disease. Autophagy receptors mediate selectivity by connecting targets to the autophagosome membrane. It has remained unknown whether receptors perform additional functions. Here, we show that in yeast certain receptor-bound targets activate Atg1, the kinase that controls autophagosome formation. Specifically, we found that in nutrient-rich conditions, Atg1 is active only in a multi-subunit complex comprising constitutive protein aggregates, their autophagy receptor, and a scaffold protein Atg11. Development of a cell-free assay for Atg1-mediated phosphorylation enabled us to activate Atg1 with purified receptorbound aggregates and Atg11. Another target, damaged peroxisomes, also activated Atg1 using Atg11 with a distinct receptor. Our work reveals that receptor-target complexes activate Atg1 to drive formation of selective autophagosomes. This regulatory logic is a key similarity between selective autophagy and bulk autophagy, which is initiated by a distinct Atg1 activation mechanism during starvation.
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    A Portrait of the GET Pathway as a Surprisingly Complicated Young Man
    (Elsevier, 2012) Denic, Vladimir
    Many eukaryotic membrane proteins have a single C-terminal transmembrane domain that anchors them to a variety of organelles in secretory and endocytic pathways. These tail-anchored (TA) proteins are post-translationally inserted into the endoplasmic reticulum by molecular mechanisms that have long remained mysterious. This review describes how, in just the past 5 years, intense research by a handful of laboratories has led to identification of all the key components of one such mechanism, the guided entry of TA proteins (GET) pathway, which is conserved from yeast to man. The GET pathway is both surprisingly complicated and yet more experimentally tractable than most other membrane insertion mechanisms, and is rapidly revealing new fundamental concepts in membrane protein biogenesis.
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    ER Targeting and Insertion of Tail-Anchored Membrane Proteins by the GET Pathway
    (Cold Spring Harbor Laboratory Press, 2013-06-14) Denic, Vladimir; Dötsch, Volker; Sinning, Irmgard
    Hundreds of eukaryotic membrane proteins are anchored to membranes by a single transmembrane domain at their C-terminus. Many of these tail-anchored (TA) proteins are post-translationally targeted to the endoplasmic reticulum (ER) membrane for insertion by the Guided-Entry of TA protein insertion (GET) pathway. In recent years most of the components of this conserved pathway have been biochemically and structurally characterized. Get3 is the pathway targeting factor that utilizes nucleotide-linked conformational changes to mediate the delivery of TA proteins between the GET pre-targeting machinery in the cytosol and the transmembrane pathway components in the ER. Here we focus on the mechanism of the yeast GET pathway and make a speculative analogy between its membrane insertion step and the ATPase-driven cycle of ABC transporters.
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    The Crystal Structures of Yeast Get3 Suggest a Mechanism for Tail-Anchored Protein Membrane Insertion
    (Public Library of Science, 2009) Hu, Junbin; Li, Jingzhi; Qian, Xinguo; Denic, Vladimir; Sha, Bingdong; Mayer, Claudine
    Tail-anchored (TA) proteins represent a unique class of membrane proteins that contain a single C-terminal transmembrane helix. The post-translational insertion of the yeast TA proteins into the ER membrane requires the Golgi ER trafficking (GET) complex which contains Get1, Get2 and Get3. Get3 is an ATPase that recognizes and binds the C-terminal transmembrane domain (TMD) of the TA proteins. We have determined the crystal structures of Get3 from two yeast species, S. cerevisiae and D. hansenii, respectively. These high resolution crystal structures show that Get3 contains a nucleotide-binding domain and a “finger” domain for binding the TA protein TMD. A large hydrophobic groove on the finger domain of S. cerevisiae Get3 structure might represent the binding site for TMD of TA proteins. A hydrophobic helix from a symmetry-related Get3 molecule sits in the TMD-binding groove and mimics the TA binding scenario. Interestingly, the crystal structures of the Get3 dimers from S. cerevisiae and D. hansenii exhibit distinct conformations. The S. cerevisiae Get3 dimer structure does not contain nucleotides and maintains an “open” conformation, while the D. hansenii Get3 dimer structure binds ADP and stays in a “closed” conformation. We propose that the conformational changes to switch the Get3 between the open and closed conformations may facilitate the membrane insertions for TA proteins.
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    The AAA protein Msp1 mediates clearance of excess tail-anchored proteins from the peroxisomal membrane
    (eLife Sciences Publications, Ltd, 2017) Weir, Nicholas R; Kamber, Roarke A; Martenson, James; Denic, Vladimir
    Msp1 is a conserved AAA ATPase in budding yeast localized to mitochondria where it prevents accumulation of mistargeted tail-anchored (TA) proteins, including the peroxisomal TA protein Pex15. Msp1 also resides on peroxisomes but it remains unknown how native TA proteins on mitochondria and peroxisomes evade Msp1 surveillance. We used live-cell quantitative cell microscopy tools and drug-inducible gene expression to dissect Msp1 function. We found that a small fraction of peroxisomal Pex15, exaggerated by overexpression, is turned over by Msp1. Kinetic measurements guided by theoretical modeling revealed that Pex15 molecules at mitochondria display age-independent Msp1 sensitivity. By contrast, Pex15 molecules at peroxisomes are rapidly converted from an initial Msp1-sensitive to an Msp1-resistant state. Lastly, we show that Pex15 interacts with the peroxisomal membrane protein Pex3, which shields Pex15 from Msp1-dependent turnover. In sum, our work argues that Msp1 selects its substrates on the basis of their solitary membrane existence.