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. 2023 May 18;136(10):jcs260578. doi: 10.1242/jcs.260578

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© 2023. Published by The Company of Biologists Ltd

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed.

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Fig. 2. — CQD1 is a negative genetic interactor of UPS1 and CRD1. (A) Deletion of CQD1 does not result in a growth defect. Cells of wild-type (WT) and a Δcqd1 deletion strain were grown to logarithmic growth phase in rich medium containing glucose as carbon source (YPD). Cell growth was analyzed by drop dilution assay on plates containing rich medium supplemented with either glucose (YPD) or glycerol (YPG) at 30°C or 37°C. (B) Schematic illustration of the mitochondrial phospholipid metabolism. OM, outer membrane; IMS, intermembrane space; IM, inner membrane; PA, phosphatidic acid; CDP-DAG, cytidine diphosphate diacylglycerol; PGP, phosphatidylglycerol phosphate; PG, phosphatidylglycerol; CL, cardiolipin; MLCL, monolysocardiolipin; PS, phosphatidylserine; PE, phosphatidylethanolamine. (C) Deletion of CQD1 from cells lacking Ups1 or Crd1 results in a synthetic growth defect. Cells were treated as in A with the difference that they were shifted to synthetic medium containing glucose (SCD) 30 h before growth analysis on SCD plates. (D) The conserved amino acids K275 and D288 in the predicted protein kinase-like domain are important for the stability of Cqd1. Cells bearing either the empty plasmid (Ø) or plasmids carrying the respective cqd1 alleles were grown in SCGal. Crude mitochondria were isolated and the Cqd1 levels were analyzed by immunoblotting using an anti-Cqd1 antibody. Upper panel, immunoblot of one representative experiment. The asterisk indicates a cross reaction of the anti-Cqd1 antibody. Lower panel, quantitative analysis of Cqd1 steady-state level in the different strains analyzed in three biological replicates. Quantification was undertaken with Image Studio software. Error bars indicate s.d. **P≤0.01; ***P≤0.001 (one-way ANOVA with subsequent Tukey's multiple comparison test). (E) Glutamic acid 330 is essential for the function of Cqd1. Cells bearing either the empty plasmid or a plasmid carrying CQD1 wild type (WT) or cqd1(E330A) alleles were treated as in C. Images in A, C and E are representative of at least three repeats.