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Science 338 (6107): 655-659

Copyright © 2012 by the American Association for the Advancement of Science

Chloroplast Biogenesis Is Regulated by Direct Action of the Ubiquitin-Proteasome System

Qihua Ling*, Weihua Huang*,{dagger}, Amy Baldwin{ddagger}, and Paul Jarvis§

Department of Biology, University of Leicester, Leicester LE1 7RH, UK.


Figure 1
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Fig. 1. The sp1 mutation suppresses the phenotype of the atToc33 knockout mutation, ppi1. (A) Plants grown on soil for 30 days. (B) Leaf chlorophyll contents of similar 40-day-old plants. (C) Ultrastructure of typical cotyledon chloroplasts in 10-day-old plants grown in vitro. Scale bar, 2 μm. These and other micrographs were used to estimate (D) chloroplast cross-sectional area and (E) thylakoid development. (F) Protein import into isolated chloroplasts was measured by quantifying maturation (mat) of in vitro translated (IVT) Rubisco small subunit precursors (pre). (G) Analysis of chloroplast proteins by SDS–polyacrylamide gel electrophoresis and SYPRO (Invitrogen, Eugene, Oregon) staining, revealing sp1-linked restoration of the three main photosynthetic proteins: Rubisco large (LSU) and small (SSU) subunits; light-harvesting chlorophyll-binding protein (LHCP). All values are means ± SEM (n ≥ 4 experiments or samples).

 

Figure 2
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Fig. 2. SP1 is located in the chloroplast outer-envelope membrane with its RING domain facing the cytosol. (A) SP1 protein map showing transmembrane (TMD), intermembrane space (SP1ims), cytosolic (SP1cyt), and RING finger (RNF) domains. (B) Localization of SP1-YFP to chloroplast envelopes (top) depended on the transmembrane domains, as revealed by a double-deletion mutant (bottom). Scale bar, 10 μm. (C) Radiolabeled SP1 in isolated chloroplasts was located in the membrane pellet (P) fraction after high-pH washing, in contrast with imported mature SSU which was in the soluble (S) fraction. Endogenous markers partitioned as expected (Coomassie stain; bottom). (D) Radiolabeled SP1 and C-terminally tagged SP1–hemagglutinin (HA)–FLAG were imported into chloroplasts before their treatment with thermolysin (Th), trypsin (Tryp), thermolysin plus Triton X-100 (Th/TX) (Fisher Scientific, Fair Lawn, New Jersey), or buffer lacking protease (Mock). Phosphor-imaging revealed protease sensitivity and protected fragments (of sizes not influenced by the C-terminal tag) consistent with outer membrane localization and the topology shown in (A). Immunoblot analysis of three endogenous markers confirmed efficacy of the treatments.

 

Figure 3
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Fig. 3. SP1 associates with TOC complexes and targets TOC components for UPS-mediated degradation via ubiquitination. (A) Immunoblot analysis of total leaf protein from different genotypes, including SP1 overexpressors (OX). Plasma membrane H+-ATPase, PMA2, acted as a loading control. Bars show means ± SEM (n = 4 to 6 experiments). (B) Coimmunoprecipitation (IP) of TOC components with HA-tagged SP1 from protoplast extracts. Cells were transfected with an SP1-HA construct or empty vector (v). (C) In vitro pull-down of radiolabeled TOC components [or domains (25)] with GST-SP1 baits. (D) In vitro ubiquitination of radiolabeled TOC components (but not atToc159G) by recombinant GST-SP1flex. Asterisks indicate a nonspecific 48-kD band seen in all translations. Mono-ubiquitinated atToc159G would be expected to migrate near the 50 kD marker. (E) Ubiquitination of atToc33 as in (D) using free and HA-tagged ubiquitin (8.5 and 9.4 kD, respectively). (F) Immunoprecipitation of TOC components with FLAG-tagged ubiquitin from transfected protoplasts. (G) Immunoprecipitation under denaturing conditions of FLAG-ubiquitin with atToc33; a control IP used excess anti-atTic110. Immunoglobulin G heavy chain (hc) is shown. Ubiquitinated species (Ub) are indicated [(D) to (G)].

 

Figure 4
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Fig. 4. SP1 is important for developmental processes that require reorganization of the plastid proteome. (A to E) De-etiolation of seedlings grown in darkness for 6 [(A) to (D)] or 5 (E) days, upon transferral to continuous light. [(A) and (B)] Cotyledons of typical plants, and survival rates, after two days of illumination. [(C) and (D)] Ultrastructure of typical cotyledon plastids after 0, 6, and 24 hours of illumination, and proportion of plastids at each of three progressively more advanced developmental stages (25) after 6 hours of illumination. Scale bar, 2 μm. (E) Chlorophyll contents after 16 hours of illumination. (F and G) Senescence of leaves induced by covering with aluminum foil. (F) Typical control (uncovered) and senescent (covered) leaves. (G) Photochemical efficiency of photosystem II (Fv/Fm) was measured to estimate the extent of senescence. All values are means ± SEM (n = 3 to 9 experiments or samples).

 


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