Journal: Cell

Article Title: Cytosolic Protein Vms1 Links Ribosome Quality Control to Mitochondrial and Cellular Homeostasis.

doi: 10.1016/j.cell.2017.10.002

Figure Lengend Snippet: Figure 2. Deletion of Vms1 and Ltn1 Causes Respiratory Deficiency (A) Growth of wild-type (WT), ltn1D, vms1D, and vms1Dltn1D cells. Cells were grown in YPD for 8 to 10 hr at 30C (to log-phase) and spotted in 10-fold dilution steps on YPD (fermentable carbon source) or YPG plates (non-fermentable carbon source). YPD plates were incubated for 3 days (23C) or for 2 days (30C and 37C) and YPG plates for 4 days (23C) or for 3 days (30C and 37C). (B) Mitochondria were isolated from WT, ltn1D, vms1D, and vms1Dltn1D cells grown in YPD at 30C. Proteins were analyzed by SDS-PAGE and immunoblotting using the antibodies indicated. (C) Cell extracts prepared with SDS-containing buffer from WT or vms1Dltn1D cells grown in YPGal medium at 23C, 30C, or 37C were analyzed by SDS-PAGE and anti-Rip1 immunoblotting. i-Rip1, intermediate and m-Rip1, mature Rip1. Phosphoglycerate kinase (Pgk1) was analyzed as loading control. (D) Mitochondria were isolated from WT, ltn1D, vms1D, and vms1Dltn1D cells grown in YPGal at 37C. Mitochondria were lysed with 3% digitonin and analyzed by blue native PAGE and immunoblotting against cytochrome c1 (Cyt1) (left) and the b subunit of F1FO-ATP synthase (F1b) (right). III and IV, complexes III and IV of the respiratory chain. See also Figure S2.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Mouse monoclonal anti-Myc Max-Planck-Institute of Biochemistry N/A Mouse anti-GFP Roche Cat. #11814460001 Mouse monoclonal anti-HA Santa Cruz biochemistry Cat. #sc-7392 Mouse monoclonal anti-Pgk1 Life Technologies Cat. #459250 Mouse monoclonal anti-ubiquitin Santa Cruz biochemistry Cat. #sc-8017 Rabbit monoclonal anti-FLAG Sigma-Aldrich Cat. #F7425 Rabbit polyclonal anti-Cytb This paper N/A Rabbit polyclonal anti-Cox2 This paper N/A Rabbit polyclonal anti-F1b This paper N/A Rabbit polyclonal anti-Rip1 This paper N/A Rabbit polyclonal anti-Tom40 This paper N/A Rabbit polyclonal anti-Tim50 This paper N/A Rabbit polyclonal anti-Tim23 This paper N/A Rabbit polyclonal anti-Mic60 This paper N/A Rabbit polyclonal anti-Mia40 This paper N/A Rabbit polyclonal anti-Cyt1 This paper N/A Rabbit polyclonal anti-Hsp60 Izawa et al., 2012 N/A Rabbit polyclonal anti-Ssc1 This paper N/A Rabbit polyclonal anti-Ssq1 This paper N/A Rabbit polyclonal anti-Ssa1 Takano et al., 2015 N/A Rabbit polyclonal anti-BiP This paper N/A Rabbit polyclonal anti-Sis1 Cosmo Bio Cat. #cop-080051 Rabbit polyclonal anti-Sis1 D. Cyr, UNC Chapel Hill N/A Rabbit anti-Cdc48 Richly et al., 2005 N/A IRDye 800CW Goat anti-Mouse IgG (H+L) LI-COR Cat. #926-32210 IRDye 800CW Goat anti-Rabbit IgG (H+L) LI-COR Cat. #926-32211 IRDye 680RD Goat anti-Mouse IgG (H+L) LI-COR Cat. #962-68070 Goat Anti-Rabbit IgG (H+L)-HRP Conjugate BIO-RAD Cat. #170-6515 Anti-Rabbit IgG (whole molecule)-Peroxidase antibody produced in goat Sigma-Aldrich Cat. #A6154 Goat anti-mouse IgG HRP conjugated Dako Cat. #P0447 Chemicals, Peptides, and Recombinant Proteins Complete protease inhibitor cocktail, EDTA-free Roche Cat. #05 056 489 001 Protein A Sepharose CL-4B GE healthcare Cat. #17-0963-03 Anti-HA Affinity Matrix Roche Cat. #11 815 016 001 Zymolyase 20T Amsbio Cat. #120491-1 Phusion High-Fidelity DNA Polymerase NEB Cat. #M0530S Q5 High-Fidelity DNA Polymerase NEB Cat. #M0491S Sequencing Grade Modified Trypsin Promega Cat. #V5111 Lysyl Endopeptidase, Mass Spectrometry Grade Wako Cat. #125-05061 Critical Commercial Assays mMACS GFP Isolation Kit Miltenyi Biotec Cat. #130-091-125 PD-10 Desalting columns GE healthcare Cat. #17-0851-01 (Continued on next page) Cell 171, 1–14.e1–e10, November 2, 2017 e1

Techniques: Incubation, Isolation, SDS Page, Western Blot, Control, Blue Native PAGE

Journal: Journal of biochemistry

Article Title: The protein N-terminal acetyltransferase A complex contributes to yeast mitophagy via promoting expression and phosphorylation of Atg32.

doi: 10.1093/jb/mvab068

Figure Lengend Snippet: Fig. 1. Loss of the NatA complex affects Atg32 phosphorylation profiles. Wild-type, ard1D and nat1D cells transformed with a plasmid encod- ing Atg32-3HA (pATG32-3HA) were grown in non-fermentable glycerol medium (Gly), collected at the indicated OD600 points, and sub- jected to western blotting. All strains are pep4- and prb1-null derivatives (defective for vacuolar degradation) lacking the endogenous ATG32 gene (atg32D (1)) or expressing Atg32-3HA from the chromosomal locus (ATG32-3HA (2)). Atg32 is phosphorylated at the early stages of respiratory growth, and phosphorylated Atg32 molecules are detected as multiple upper bands. Arrowheads indicate putative phosphory- lated Atg32. Pgk1 was monitored as a loading control.

Article Snippet: After treatment with the horseradish peroxidase-conjugated rabbit anti-mouse IgG (HþL) secondary antibody for mCherry, GFP, HA and Pgk1 (1:10,000; Jackson ImmunoResearch, 315-035- 003) followed by the enhanced chemiluminescence reagent ChemiLumi One L (Nacalai, 07880-70) or Western Lightning Plus-ECL (PerkinElmer, NEL105001EA), proteins were detected using a luminescent image analyzer (FUSION Solo S; Vilber).

Techniques: Phospho-proteomics, Transformation Assay, Plasmid Preparation, Western Blot, Expressing, Control

Journal: Journal of biochemistry

Article Title: The protein N-terminal acetyltransferase A complex contributes to yeast mitophagy via promoting expression and phosphorylation of Atg32.

doi: 10.1093/jb/mvab068

Figure Lengend Snippet: Fig. 2. Hyperphosphorylation of Atg32 partially rescues mitophagy defects in NatA-deficient cells. (A, B) Wild-type, ppg1D, ard1D, ard1D ppg1D, nat1D, nat1D ppg1D, and atg32D cells expressing mitochondria-targeted DHFR-mCherry (mito-DHFR-mCherry) were pregrown to mid-log phase in glucose medium (Gly 0 h), cultured in glycerol medium (Gly), collected at the indicated time points, and subjected to west- ern blotting. Generation of free mCherry indicates transport of mitochondria to the vacuole. Free mCherry signals in cells at the indicated time points were quantified more than three times in independent experiments. The signal intensity of free mCherry in wild-type cells at the 48 h time point was set to 100%. Data represent the averages of all experiments, with bars indicating standard deviations. (C, D) Wild-type, ard1D, and nat1D cells expressing full-length Atg32-3HA (ATG32-3HA), an Atg32 deletion mutants (D151–200) fused with 3HA ((D151– 200)-3HA) or not expressing Atg32 were pregrown to mid-log phase in glucose medium (Gly 0 h), cultured in glycerol medium (Gly), col- lected at the indicated time points, and subjected to western blotting. This deletion mutant is highly phosphorylated to strongly promote mitophagy. All strains are atg32-null derivatives (atg32D) expressing mito-DHFR-mCherry. Free mCherry signals in cells at the indicated time points were quantified more than three times in independent experiments. The signal intensity of free mCherry in wild-type cells at the 48 h time point was set to 100%. Data represent the averages of all experiments, with bars indicating standard deviations. (E) Wild-type, ppg1D, ard1D, ard1D ppg1D, nat1D, nat1D ppg1D, and atg32D cells expressing Atg32-3HA were grown in glycerol medium (Gly), collected at the indicated OD600 points, and subjected to western blotting. All strains are derivatives lacking Atg7, a protein essential for all autophagy- related processes, to avoid degradation of Atg32-3HA via mitophagy. Atg32-3HA signals normalized with Pgk1 (loading control) signals were quantified more than three times in independent experiments. Data represent the averages of all experiments, with bars indicating standard deviations. *Non-specific bands. (F) Wild-type, ard1D, and nat1D cells expressing full-length Atg32-3HA (ATG32-3HA), an Atg32 deletion mutants (D151–200) fused with 3HA ((D151–200)-3HA), or not expressing Atg32 were grown in glycerol medium (Gly), collected at the indicated OD600 points, and subjected to western blotting. All strains are atg7-null derivatives. Atg32-3HA signals normalized with Pgk1 (loading control) signals were quantified more than three times in independent experiments. Data represent the averages of all experiments, with bars indicating standard deviations. *Non-specific bands.

Article Snippet: After treatment with the horseradish peroxidase-conjugated rabbit anti-mouse IgG (HþL) secondary antibody for mCherry, GFP, HA and Pgk1 (1:10,000; Jackson ImmunoResearch, 315-035- 003) followed by the enhanced chemiluminescence reagent ChemiLumi One L (Nacalai, 07880-70) or Western Lightning Plus-ECL (PerkinElmer, NEL105001EA), proteins were detected using a luminescent image analyzer (FUSION Solo S; Vilber).

Techniques: Expressing, Cell Culture, Western Blot, Mutagenesis, Control

Journal: Molecular cell

Article Title: Ribosome collision is critical for quality control during no-go decay

doi: 10.1016/j.molcel.2017.08.019

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Mouse monoclonal anti-PGK1[22C5D8] , Abcam , Cat#: ab113687; RRID:AB_10861977.

Techniques: Virus, Recombinant, Reverse Transcription, SYBR Green Assay, Random Hexamer, Luciferase, Reporter Assay, PCR Cloning, Ligation, Cloning, Plasmid Preparation, Software