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. 2014 May;26(5):2201-2222.
doi: 10.1105/tpc.114.124842. Epub 2014 May 30.

Conditional Depletion of the Chlamydomonas Chloroplast ClpP Protease Activates Nuclear Genes Involved in Autophagy and Plastid Protein Quality Control

Silvia Ramundo  1 , David Casero  2 , Timo Mühlhaus  3 , Dorothea Hemme  3 , Frederik Sommer  3 , Michèle Crèvecoeur  1 , Michèle Rahire  1 , Michael Schroda  3 , Jannette Rusch  4 , Ursula Goodenough  4 , Matteo Pellegrini  5 , Maria Esther Perez-Perez  6 , José Luis Crespo  6 , Olivier Schaad  7 , Natacha Civic  8 , Jean David Rochaix  9
Affiliations

Conditional Depletion of the Chlamydomonas Chloroplast ClpP Protease Activates Nuclear Genes Involved in Autophagy and Plastid Protein Quality Control

Silvia Ramundo et al. Plant Cell. 2014 May.

Abstract

Plastid protein homeostasis is critical during chloroplast biogenesis and responses to changes in environmental conditions. Proteases and molecular chaperones involved in plastid protein quality control are encoded by the nucleus except for the catalytic subunit of ClpP, an evolutionarily conserved serine protease. Unlike its Escherichia coli ortholog, this chloroplast protease is essential for cell viability. To study its function, we used a recently developed system of repressible chloroplast gene expression in the alga Chlamydomonas reinhardtii. Using this repressible system, we have shown that a selective gradual depletion of ClpP leads to alteration of chloroplast morphology, causes formation of vesicles, and induces extensive cytoplasmic vacuolization that is reminiscent of autophagy. Analysis of the transcriptome and proteome during ClpP depletion revealed a set of proteins that are more abundant at the protein level, but not at the RNA level. These proteins may comprise some of the ClpP substrates. Moreover, the specific increase in accumulation, both at the RNA and protein level, of small heat shock proteins, chaperones, proteases, and proteins involved in thylakoid maintenance upon perturbation of plastid protein homeostasis suggests the existence of a chloroplast-to-nucleus signaling pathway involved in organelle quality control. We suggest that this represents a chloroplast unfolded protein response that is conceptually similar to that observed in the endoplasmic reticulum and in mitochondria.

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Figures

Figure 1.
Figure 1.
Characterization of the C. reinhardtii DCH16 Strain upon ClpP1 Depletion. (A) Growth patterns of the Rep112, A31, and DCH16 strains. Cells were spotted on HSM (minimal medium) and TAP in the absence or presence (+vit) of vitamins B12 (20 μg/L) and thiamine (20 μM). Irradiance was 10 μmol m−2 s−1. (B) Growth curves of the A31and DCH16 strains after addition of vitamins. To maintain cells in exponential growth during the entire time course, they were diluted to 0.5 × 106 cells/mL when they reached a concentration between 2 and 4 × 106 cells/mL. The experiment was repeated three times with similar results. (C) Decrease of PSII activity in DCH16 after addition of vitamins. Fv/Fm was measured for the indicated strains at different times after addition of vitamins. The experiment was repeated three times with similar results. (D) RNA gel blot analysis of chloroplast genes in DCH16, A31, and ClpP1-AUU in the presence of vitamins or rapamycin. RNA was extracted from the strains at different time points after addition of vitamins and hybridized with the indicated probes. ClpP1-AUU is the strain in which the AUG initiation codon of ClpP1 was replaced by AUU. Arrows point to the different transcripts of ORF1995, a chloroplast gene of unknown function. (E) RNA gel blot analysis of nuclear genes. Strains and conditions were as in (D). Rapamycin treated cells were transferred from 10 to 60 μmol photons m−2 s−1 upon addition of the drug. vit, vitamins; Rap, rapamycin.
Figure 2.
Figure 2.
Immunoblot Analysis of Chloroplast Proteins in DCH16, A31, and ClpP1-AUU in the Presence of Vitamins or Rapamycin. (A) Immunoblot analysis. Proteins were extracted from the three strains grown under an irradiance of 10 μmol m−2 s−1 at different time points after addition of vitamins and reacted with antibodies directed against the indicated proteins as shown to the left of the immunoblot. (B) Analysis of proteins from DCH16 grown in the light or the dark upon ClpP1 repression. Proteins were extracted at different time points and immunoblotted with the antisera as indicated to the left of the immunoblot. Irradiance was 60 μmol m−2 s−1.
Figure 3.
Figure 3.
Immunoblot Analysis during ClpP1 Depletion. (A) Immunoblot analysis. Proteins were extracted from the three indicated strains grown under an irradiance of 10 μmol m−2 s−1 at different time points after addition of vitamins and reacted with antibodies directed against specific proteins. (B) Changes in partitioning of Vipp1 during ClpP1 depletion. DCH16 cells were grown in the presence of vitamins in the dark. After cell lysis, cell extracts of DCH16 were separated into soluble and insoluble fractions by centrifugation and subjected to gel electrophoresis and immunoblotting with the indicated antibodies. (C) Total cell extracts from DCH16 were subjected to gel electrophoresis in the presence of SDS without (left panel) and with urea (right panel). Cells were grown in the dark or with an irradiance of 60 μmol m−2 s−1. The smear at the top of the gel marked with asterisks represents aggregated protein. The same amount of protein was loaded in each lane.
Figure 4.
Figure 4.
Increased Vacuolization of DCH16 upon Depletion of ClpP1. (A) Analysis by transmission electron microscopy of epoxy-embedded thin sections of DCH16 and A31 cells following ClpP1 repression. Cells grown in the light (L; 60 μmol m−2 s−1) or in the dark (D) were examined at different times as indicated. Dark spots represent polyphosphate granules (Komine et al., 2000). Bars = 2 μm. (B) Analysis by light microscopy of DCH16 grown in the absence (left panel) or presence of vitamins (right panel) for 4 d. L, light; V, vacuole; N, nucleus. Bars = 10 μm.
Figure 5.
Figure 5.
Quick-Freeze Deep-Etch Electron Micrographs of DHC16 Cells. Following vitamin addition in dim light (30 μmol m−2 s−1), images were taken after 0 ([A] to [C]), 48 ([D] to [F]), and 72 h ([G] and [H]). In (E), perforations in the membrane are indicated by small arrows. AV, autophagocytic vacuole; C, chloroplast; ce, chloroplast envelope; cv, contractile vacuole vesicles; G, Golgi; N, nucleus; pm, plasma membrane; S, starch granules; V, resting vacuole; WV, water-filled vacuole. Bars = 500 nm.
Figure 6.
Figure 6.
Effect of Rapamycin on Cell Morphology. (A) Electron microscope analysis of A31 cells treated for 8 h with rapamycin (RAP). Bars = 2 μm. (B) Induction of Atg8 upon ClpP1 depletion. Immunofluorescence with an Atg8 antiserum was performed on DCH16 cells grown for 48 h in the absence (+ClpP1) or presence of vitamins (-ClpP1). (C) Induction of Atg8 upon ClpP1 depletion. Immunofluorescence with an Atg8 antiserum was performed on DCH16 cells grown for 72 h with vitamins. Cells were visualized in a differential interference contrast microscope. (D) Induction of lipids upon ClpP1 depletion. Left panels: Staining of DCH16 cells grown in the absence or presence of vitamins with Nile red. Right panels: Staining of DCH16 cells grown in the absence or presence of rapamycin with Nile red. L, light (60 μmol m−2 s−1); DL, dim light (10 μmol m−2 s−1). Bars = 4 μm in (B) to (D).
Figure 7.
Figure 7.
Model-Based Clustering of RNA-Seq Data. Groups of genes with similar expression profiles across all samples were obtained using a negative binomial model as described (Si et al., 2014). (A) Heat map of expression levels for significantly regulated genes (a total of 5085; see main text). The map shows the fold change at 0, 12, 31, 43, and 48 h following vitamin addition for the A31 and DCH16 strains (from bottom to top of the heat map), using the gene's average expression as a reference. The color scale ranges from dark red (positive fold changes) to illustrate significant overexpression, to light yellow (negative fold changes). The tree shows the result of the hybrid-hierarchical clustering, which groups clusters based on their similarity (as computed from the likelihood of the negative binomial model). The lower level of the tree and labels on the x axis represent the maximum number of clusters used in this work (12). (B) Kernel-density line plots for individual clusters (from 1 to 12). Each plot compiles the expression profiles for both strains (A31 and DCH16) across the time course (0, 12, 31, 43, and 48 h). For each cluster, the red line shows the cluster’s trend (average expression profile of all genes in the cluster), and the light-blue density map is a histogram of all the individual gene profiles. Also shown are the most significant functional ontologies as described in the main text.
Figure 8.
Figure 8.
Specific Expression of Nuclear Genes upon ClpP1 Depletion in DCH16. (A) A construct with the VIPP2 promoter and 5′ UTR fused to the Gaussia luciferase gene (proVipp2-gLUX) was introduced into DCH16 (CS191 strain). Immunoblots were performed with total cell extracts from CS191 grown for the times indicated in the presence of vitamins in the light (60 μmol m−2 s−1) or in the dark as indicated using antibodies against ClpP1, luciferase (gLUX), Vipp2, Vipp1, and RbcL. These cells were also treated with rapamycin (Rap), tunicamycin (Tm), and high light (HL; 600 μmol m−2 s−1) or grown in the absence of nitrogen (-N) . The times of each treatment are indicated. (B) A construct with the Deg11 promoter and 5′ UTR fused to the Gaussia luciferase gene (proDeg11-gLUX) was introduced into DCH16 (CS 174 strain). Immunoblot analysis of extracts from CS174 was performed as in (A). Rapamycin-treated cells were grown under constant light (60 μmol m−2 s−1).
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