Biogenic membranes of the chloroplast in Chlamydomonas reinhardtii

Abstract

The polypeptide subunits of the photosynthetic electron transport complexes in plants and algae are encoded by two genomes. Nuclear genome-encoded subunits are synthesized in the cytoplasm by 80S ribosomes, imported across the chloroplast envelope, and assembled with the subunits that are encoded by the plastid genome. Plastid genome-encoded subunits are synthesized by 70S chloroplast ribosomes directly into membranes that are widely believed to belong to the photosynthetic thylakoid vesicles. However, in situ evidence suggested that subunits of photosystem II are synthesized in specific regions within the chloroplast and cytoplasm of Chlamydomonas. Our results provide biochemical and in situ evidence of biogenic membranes that are localized to these translation zones. A "chloroplast translation membrane" is bound by the translation machinery and appears to be privileged for the synthesis of polypeptides encoded by the plastid genome. Membrane domains of the chloroplast envelope are located adjacent to the cytoplasmic translation zone and enriched in the translocons of the outer and inner chloroplast envelope membranes protein import complexes, suggesting a coordination of protein synthesis and import. Our findings contribute to a current realization that biogenic processes are compartmentalized within organelles and bacteria.

Fig. 1.

Chloroplast translation membranes were revealed by subcellular fractionation. Each panel shows the results from an independent trial of our subcellular fractionation scheme. Fractions were assayed with immunoblots for the following marker proteins: appressed (granal) thylakoid membranes (D2), stroma-exposed thylakoid membranes (PsaAp), CTM and the T-zone (RBP40, r-proteins of the 30S and 50S chloroplast ribosomal subunits), the TOC-TIC protein translocons of the outer and inner chloroplast envelope membranes (Toc75 and Tic110, respectively), chloroplast stroma (HSP70B), PSII assembly (YFCF48/HCF136), PSI assembly (YCF4p), and the cytoplasmic ribosome (60S). (A) Percentages of total chlorophyll (%Chl) and protein (%Prot) in each fraction are indicated. The supernatant of the initial high speed centrifugation is labeled “S.” Membranes of the sucrose gradient were collected as (A) fractions 1–13 or (B and C) fractions 1–12. The 2.5 M sucrose from which membranes were floated is (A) fraction 14 and (B and C) fraction 15. The pellet of the sucrose gradient (P). A thin line in each row distinguishes the images of immunoblots of two gels for which all steps were carried out in the same solutions and ECL and photographic exposures.

Fig. 2.

CTM association of ribosome subunits and RBP40. Samples of CTM (fraction 10 in Fig. 1B) were incubated with the indicated agents to extract peripheral membrane proteins. Membranes were pelleted by centrifugation and then immunoblot analyses compared the nonmembrane supernatant (S) and membrane pellet (P) fractions to reveal the degrees of extraction of RBP40 and the 30S and 50S subunits of the chloroplast ribosome. The trace amount of thylakoid membranes in this sample allowed us to ensure pelleting of membranes by immunoprobing for D2.

Fig. 3.

BN-PAGE revealed markers of protein synthesis for PSII de novo assembly and repair. (A) Analysis by 1D BN-PAGE compared the assembly states of D2 in thylakoids (lanes 1–2) and CTM (lanes 3–6); samples of fractions 8–13 in Fig. 1A. D2 was immunodetected in RCC1, RCC2, and RC47. Samples were normalized to the level of RCC1 to ensure comparable solubilization conditions (Results). (B–G) To reveal subcomplexes and unassembled free subunits, BN-PAGE lanes with thylakoid membranes or CTM, equivalent to lanes 1 and 6 in A, respectively, were subjected to a second dimension of SDS/PAGE before immunoblot analyses. RCC1 levels determined in A were used to normalize samples analyzed on the 2D gels. The 2D gel-immunoblots of thylakoid membranes (B, D, and F) or CTM (C, E, and G) were first immuno-probed for D1 (B and C), then for D2 (D and E), and finally for the PSI subunit PsaAp (F and G). D1 and D2 were detected in RCC1, RCC2, and RC47, in smaller assembly intermediate precomplexes [RC47 and PSII reaction center (RC)] and as unassembled subunits (UP). The expected molecular mass of each protein is indicated by an asterisk. Some D1 and D2 was shifted to higher molecular mass positions of the gels (**) because of incomplete denaturation before the second dimension of SDS/PAGE. This shift was useful because it resolved the RC (shifted*) from the free subunits (not shifted**). The same results were obtained when this shift did not occur. (F and G) PsaAp was detected in the PSI monomer (PSI), a putative PSI monomer lacking PsaK and PsaG (PSI-PsaK/G), and an unknown complex, possibly the PSI dimer (“?”).

Fig. 4.

The TOC and TIC protein import complexes are localized to chloroplast envelope domains. (A) An illustration of a Chlamydomonas cell shows the nucleus (N), cytosol, and chloroplast with its lobes, lobe junctions, basal region, thylakoid lamellae, T-zone, and pyrenoid (P). The chloroplast lobes extend from the basal region to the anterior cell pole, thereby “cupping” the nuclear-cytosolic compartments. (B–E) Representative cells are oriented as in A and show the IF signal from Toc75 (B and C) or Tic110 (D and E). Costaining for the psbA mRNA by FISH (green) revealed the T-Zone (thin arrows). Cells in B and D (moderate light) show the localization of the Toc75 or Tic110 IF signal at lobe junctions, and cells in C and E (dark-adapted) do not show this localization pattern. (Scale bars, 2 μm.)

Fig. 5.

A working model for the spatiotemporal organization of PSII-LHCII supercomplex biogenesis. (1) In the T-zone, plastid-encoded subunits are synthesized into CTM. (LDM might represent a mRNA-ribosome subunit recruitment membrane on the far left.) (2) Free subunits assemble to form the PSII reaction center (RC) and the other precomplexes, and then (3) move by lateral diffusion to a lobe junction (see also Fig. 4A). (4) There, precomplexes associate to form the PSII monomeric complex, RCC1. (5) RCC1 dimerizes to form RCC2. (6) Nuclear genome-encoded subunits of the OEC (blue) and LHCII (light green) are locally imported by the TOC and TIC complexes (purple) into the lobe junction and assembled upon RCC1 and RCC2. (7) The resulting PSII-LHCII supercomplex diffuses to thylakoid membrane located throughout the chloroplast.