1. An Essential Role for COPI in mRNA Localization to Mitochondria and Mitochondrial Function 
Dmitry Zabezhinsky, Boris Slobodin, Doron Rapaport, Jeffrey E. Gerst 
Cell Reports 
Volume 15, Issue 3, Pages (April 2016)
DOI: /j.celrep
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2. Cell Reports 2016 15, 540-549DOI: (10.1016/j.celrep.2016.03.053)
Copyright © 2016 The Authors Terms and Conditions

3. Figure 1
COPI Subunit Inactivation Reduces OXA1 mRNA Localization to Mitochondria
(A) Endogenous OXA1 mRNA is mislocalized upon sec26-2 inactivation. WT (W303) and sec26-2 cells expressing MTS-GFP (Mito) were either maintained at 26°C or shifted to 37°C for 1 hr, prior to labeling with smFISH probes. Merge, merge of fluorescence windows. Scale bars represent 2 μm.
(B) Histogram of OXA1 smFISH data.
(C) Exogenous OXA1 mRNA is mislocalized upon COPI inactivation. WT (BY4741) and indicated COPI ts strains expressing MS2-tagged OXA1 and MS2-CP-GFP(x3) from CEN plasmids were grown as in (A), prior to staining with MitoTracker. Histogram shows the percentage of mislocalized RNA granules in cells grown at 26°C (gray bars) or shifted to 37°C (white bars). See the fold change table at the bottom.
(D) Endogenous MS2-tagged OXA1 mRNA is mislocalized upon COPI inactivation. WT (BY4741) and sec27-1 strains expressing endogenous MS2-tagged OXA1 and MS2-CP-GFP(x3) were grown as in (A) in medium containing 3% glycerol, prior to labeling with MitoTracker.
(E) mMPs are depleted from a mitochondrial fraction upon COPI inactivation. sec26-2 cells, grown as in (A), were subjected to mitochondrial fractionation, RNA extraction, and qPCR. Shown are the amounts of the indicated RNAs bound to the mitochondrial-enriched fraction derived from sec26-2 cells at 26°C or shifted to 37°C, relative to those derived from WT cells.
Error bars represent SE). ∗p ≤ 0.05; ∗∗p ≤ 0.01; ∗∗∗p ≤
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4. Figure 2 COPI Inactivation Mislocalizes OXA1 mRNA to the ER
(A) COPI inactivation leads to co-localization of endogenous OXA1 mRNA with ER. WT (W303) and sec26-2 cells expressing the Sec63-GFP ER marker were maintained at 26°C or shifted to 37°C (1 hr), prior labeling with smFISH probes.
(B) Co-localization of exogenous OXA1 mRNA with ER is enhanced upon COPI inactivation. sec33-1 and sec26-2 cells expressing MS2-tagged OXA1 mRNA, MS2-CP-GFP(x3), and the mCh-Scs2 ER marker from plasmids were grown as in (A) and scored for RNA granule localization to ER. ∗p ≤ 0.05.
(C) OXA1 mRNA mislocalization is not affected by CHX. sec26-2 cells expressing MS2-tagged OXA1 and MS2-CP-GFP(x3) from plasmids were treated with CHX (100 μg/ml) and grown as in (A). Cells were labeled with MitoTracker, and the percentage of mislocalized RNA granules was scored. ∗∗p ≤ 0.01.
(D) Sec63 fused with GFP and tandem dimers of MS2 binding protein is ER localized. WT cells expressing Sec63-GFP or Sec63-GFP fused to two copies of the MS2 aptamer-binding protein (Sec63-MCP-GFP), as illustrated, were visualized by fluorescence (GFP) and phase-contrast microscopy (Light). Scale bars represent 2 μm.
(E) Expression of ER-anchored MS2 aptamer-binding protein inhibits the growth of MS2 aptamer-tagged OXA1 cells. WT and oxa1Δ cells expressing MS2-tagged OXA1 (OXA1MS2) from a plasmid and either Sec63-GFP or Sec63-MCP-GFP were serial diluted, plated onto glucose- or glycerol-containing synthetic medium, and grown (48 hr).
Error bars represent SEM.
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5. Figure 3 COPI Inactivation Mislocalizes Oxa1 Protein to the ER
(A) Oxa1-RFP protein localizes to mitochondria in WT cells. WT cells expressing MS2 aptamer-tagged OXA1-RFP and either MTS-GFP (Mito) or GFP-tagged Scs2 (ER) from plasmids were maintained at 26°C or shifted to 37°C (1 hr) prior to visualization.
(B) Oxa1-RFP protein co-localizes with ER upon sec26-2 inactivation. sec26-2 cells were treated as in (A).
(C) Oxa1 protein co-localizes with ER upon COPI inactivation. WT and indicated COPI ts strains expressing MS2 aptamer-tagged OXA1-RFP and GFP-Scs2 were grown and analyzed as in (A). Gray bars indicate cells grown at 26°C; black bars indicate cells shifted to 37°C (1 hr). glu, medium containing glucose.
(D) Oxa1 localizes to ER in BFA-treated cells. BFA-permeable yeast (ise1Δ) expressing MS2 aptamer-tagged OXA1-RFP and GFP-Scs2 were left untreated (-) or treated with (+) BFA (100 μg/ml) for 2 hr at 26°C. Cells were analyzed for Oxa1-RFP localization to ER.
Error bars represent SEM. Scale bars represent 2 μm. ∗p ≤ 0.05; ∗∗p ≤ 0.01; ∗∗∗p ≤
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6. Figure 4 COPI Inactivation Inhibits MP Import and Respiration
(A) Import in vitro of Cyb2 and Oxa1 is inhibited upon COPI inactivation. CYB2 and OXA1 RNAs were translated in vitro using [35S]-methionine in the presence (+) or absence (-) of mitochondria (Mito) isolated from WT and sec26-2 cells grown on glycerol-containing medium and shifted to 37°C for 2 hr. m, mature form; im, immature form. Upper panels: autoradiograms of labeled proteins after SDS-PAGE; lower panels: Western blots detected with anti-Tom40 (loading control for mitochondrial fraction).
(B) Import in vitro of Cyb2 and Oxa1 is inhibited upon COPI inactivation. Histograms of mature Cyb2 (average ± SD; n = 3; p < 0.01) and Oxa1 (n = 2; p < 0.001) in sec26-2 cells, relative to WT cells, are shown.
(C) COPI function is necessary for respiration. Indicated WT, COPI ts, and galactose-inducible COPI (GAL-SEC28) strains were grown on medium containing glucose or galactose (for GAL-SEC28 cells), diluted serially (10-fold each), spotted onto solid medium containing glucose (Glu) or glycerol (Gly), and grown at the indicated temperatures for 48 hr (for Glu) or 96 hr (for Gly).
(D) MTS translation and the 3′ UTR target OXA1 mRNA to mitochondria. A schematic summarizing the MS2-tagged constructs tested and corresponding RNA localization data are shown. RNA localization to mitochondria at 26°C was scored in WT cells expressing the full-length coding sequence (CDS) and 3′ UTR (Full); and variants lacking the 3′ UTR (-3′ UTR), first initiation codon (-ATG), encoded MTS (-MTS), and both MTS and 3′ UTR (-MTS,-3′ UTR). The bottom-most construct, which contains the CDS and 3′ UTR but lacks an encoded transmembrane region (-TMD), was expressed in sec26-2 cells shifted (1 hr) to 37°C. Percentage of RNA granules localized to mitochondria was scored: +++++ indicates ≥90% co-localization; ++++ indicates ≥70%; +++ indicates≥50%; ++ indicates≥30%; and + indicates ≥10%, or background).
(E) A TMD region targets OXA1 RNA to ER. WT yeast expressing an Oxa1 TMD region with or without an initiation codon, e.g., TMD or TMD (−ATG), respectively; a corresponding region downstream of the TMD, ORF (control); or the MS2 aptamer repeats alone, MS2 (control), were scored for RNA localization to the ER. Scoring is as in (D).
See also Figure S4D.
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7. Figure 5 COPI Inactivation Affects Sec21 or Sec27 Binding of mMPs
(A) Plot of native and mutated OXA1 sequence. The potential RNA-protein interaction scores for native and mutated OXA1 are shown.
(B) Alteration of the OXA1 sequence reduces RNA binding to Sec27. SEC27-HA(x3) oxa1Δ cells expressing native or mutated OXA1 from plasmids were processed for IP and RT-PCR using oligonucleotides against OXA1 or ACT1 (control). TCL, total cell lysate.
(C) Alteration of the OXA1 sequence alone affects respiration-dependent growth. WT, oxa1Δ, and cells expressing mutated OXA1 (OXA1mut) from its genomic locus were diluted serially, plated onto glucose- or glycerol-containing medium, and grown for 48 and 72 hr, respectively.
(D) sec26-2 inactivation inhibits mMP binding by Sec21. WT and sec26-2 cells expressing Sec21-HA(x3) from the genome were maintained at 26°C or shifted to 37°C (1 hr) and processed for IP, RNA extraction, and detection of FIS1 RNA by qPCR. Error bars represent SEM.
(E) cis- and trans-acting elements regulate mRNA and protein targeting to mitochondria. mMP (e.g., OXA1) fate is regulated by cis-acting RNA elements, such as the encoded MTS (MTSRNA) and 3′ UTR, which facilitate localization to the mitochondria upon translation and when the trans-acting COPI complex is active. In contrast, when the MTS is absent, translation blocked, or COPI inactive, then mMPs localize to the ER via other cis-acting elements (e.g., the encoded TMD) in a translation-independent fashion. Undetermined trans-acting factors likely retain mMPs at the ER under these conditions. MP import into the ER may represent a dead-end path utilized during switching from non-fermentable to fermentable carbon sources.
Cell Reports  , DOI: ( /j.celrep )
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