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Sökning: WFRF:(Khmelinskii Anton)

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1.
  • Khmelinskii, Anton, et al. (författare)
  • Protein quality control at the inner nuclear membrane
  • 2014
  • Ingår i: Nature. - : Springer Science and Business Media LLC. - 0028-0836 .- 1476-4687. ; 516:7531, s. 410-
  • Tidskriftsartikel (refereegranskat)abstract
    • The nuclear envelope is a double membrane that separates the nucleus from the cytoplasm. The inner nuclear membrane (INM) functions in essential nuclear processes including chromatin organization and regulation of gene expression(1). The outer nuclear membrane is continuous with the endoplasmic reticulum and is the site of membrane protein synthesis. Protein homeostasis in this compartment is ensured by endoplasmic-reticulum-associated protein degradation (ERAD) pathways that in yeast involve the integral membrane E3 ubiquitin ligases Hrd1 and Doa10 operating with the E2 ubiquitin-conjugating enzymes Ubc6 and Ubc7 (refs 2, 3). However, little is known about protein quality control at the INM. Here we describe a protein degradation pathway at the INM in yeast (Saccharomyces cerevisiae) mediated by the Asicomplex consisting of the RING domain proteins Asi1 and Asi3 (ref. 4). We report that the Asi complex functions together with the ubiquitin-conjugating enzymes Ubc6 and Ubc7 to degrade soluble and integral membrane proteins. Genetic evidence suggests that the Asi ubiquitin ligase defines a pathway distinct from, but complementary to, ERAD. Using unbiased screening with a novel genome-wide yeast library based on a tandem fluorescent protein timer(5), we identify more than 50 substrates of the Asi, Hrd1 and Doa10 E3 ubiquitin ligases. We show that the Asi ubiquitin ligase is involved in degradation of mislocalized integral membrane proteins, thus acting to maintain and safeguard the identity of the INM.
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2.
  • Khmelinskii, Anton, et al. (författare)
  • Seamless gene tagging by endonuclease-driven homologous recombination.
  • 2011
  • Ingår i: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 6:8
  • Tidskriftsartikel (refereegranskat)abstract
    • Gene tagging facilitates systematic genomic and proteomic analyses but chromosomal tagging typically disrupts gene regulatory sequences. Here we describe a seamless gene tagging approach that preserves endogenous gene regulation and is potentially applicable in any species with efficient DNA double-strand break repair by homologous recombination. We implement seamless tagging in Saccharomyces cerevisiae and demonstrate its application for protein tagging while preserving simultaneously upstream and downstream gene regulatory elements. Seamless tagging is compatible with high-throughput strain construction using synthetic genetic arrays (SGA), enables functional analysis of transcription antisense to open reading frames and should facilitate systematic and minimally-invasive analysis of gene functions.
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