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Complete maturation of the plastid protein translocation channel requires a type I signal peptidase.

Inoue K, Baldwin AJ, Shipman RL, Matsui K, Theg SM, Ohme-Takagi M - J. Cell Biol. (2005)

Bottom Line: Next, we show that disruption of a gene encoding plastidic SPase I (Plsp1) resulted in the accumulation of immature forms of Toc75, severe reduction of plastid internal membrane development, and a seedling lethal phenotype.These phenotypes were rescued by the overexpression of Plsp1 complementary DNA.Plsp1 appeared to be targeted both to the envelope and to the thylakoidal membranes; thus, it may have multiple functions.

View Article: PubMed Central - PubMed

Affiliation: Department of Plant Sciences, College of Agricultural and Environmental Sciences, University of California, Davis, CA 95616, USA. kinoue@ucdavis.edu

ABSTRACT
The protein translocation channel at the plastid outer envelope membrane, Toc75, is essential for the viability of plants from the embryonic stage. It is encoded in the nucleus and is synthesized with a bipartite transit peptide that is cleaved during maturation. Despite its important function, the molecular mechanism and the biological significance of the full maturation of Toc75 remain unclear. In this study, we show that a type I signal peptidase (SPase I) is responsible for this process. First, we demonstrate that a bacterial SPase I converted Toc75 precursor to its mature form in vitro. Next, we show that disruption of a gene encoding plastidic SPase I (Plsp1) resulted in the accumulation of immature forms of Toc75, severe reduction of plastid internal membrane development, and a seedling lethal phenotype. These phenotypes were rescued by the overexpression of Plsp1 complementary DNA. Plsp1 appeared to be targeted both to the envelope and to the thylakoidal membranes; thus, it may have multiple functions.

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Disruption of PLSP1 results in a seedling lethal phenotype and reduction of the development of plastid internal membranes. (A) Structure of PLSP1. Gray boxes and solid lines indicate exons and introns, respectively. The position of the T-DNA insertion in plsp1-1 was determined experimentally and is indicated with a white arrowhead. Positions of primers used for genomic and RT-PCR are indicated with black arrows. Primers and inserted T-DNA indicated with arrows/arrowhead are not to scale. (B) Seedlings of wild-type (wt), plsp1-1, and plsp1-1;PLSP1 plants grown on MS media supplemented with 1% sucrose for 2 wk. Bar, 1 cm. (C) Genomic PCR of wild-type and mutant A. thaliana seedlings. E, I, and C indicate reactions specific to amplify endogenous PLSP1, the T-DNA insertion, and the transgene used to complement the mutation, respectively. Primers that were used are listed as follows: F2 and R1 for E, F2 and R3 for I, and primers from 35S promoter (forward) and nopaline synthase terminator (reverse) for C. (D) RT-PCR analyses of wild-type and mutant A. thaliana seedlings. Each reaction contained two sets of primers: one for PLSP1 cDNA (F1 and R2) that produces a 400-bp fragment and another for cDNA derived from 18S RNA that produces a 315-bp fragment. Images from different portions of the same gel are in separate boxes. (E and F) Plastids in 2-wk-old wild-type (E) and homozygous plsp1-1 (F) cotyledons. Plastoglobules are indicated with asterisks. (G and H) Plastid in 7-wk-old wild-type (G) and homozygous plsp1-1 (H) etiolated seedlings. White and black arrowheads indicate PLB and disorganized membrane structure, respectively. (E–H) Bars, 1 μm.
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fig2: Disruption of PLSP1 results in a seedling lethal phenotype and reduction of the development of plastid internal membranes. (A) Structure of PLSP1. Gray boxes and solid lines indicate exons and introns, respectively. The position of the T-DNA insertion in plsp1-1 was determined experimentally and is indicated with a white arrowhead. Positions of primers used for genomic and RT-PCR are indicated with black arrows. Primers and inserted T-DNA indicated with arrows/arrowhead are not to scale. (B) Seedlings of wild-type (wt), plsp1-1, and plsp1-1;PLSP1 plants grown on MS media supplemented with 1% sucrose for 2 wk. Bar, 1 cm. (C) Genomic PCR of wild-type and mutant A. thaliana seedlings. E, I, and C indicate reactions specific to amplify endogenous PLSP1, the T-DNA insertion, and the transgene used to complement the mutation, respectively. Primers that were used are listed as follows: F2 and R1 for E, F2 and R3 for I, and primers from 35S promoter (forward) and nopaline synthase terminator (reverse) for C. (D) RT-PCR analyses of wild-type and mutant A. thaliana seedlings. Each reaction contained two sets of primers: one for PLSP1 cDNA (F1 and R2) that produces a 400-bp fragment and another for cDNA derived from 18S RNA that produces a 315-bp fragment. Images from different portions of the same gel are in separate boxes. (E and F) Plastids in 2-wk-old wild-type (E) and homozygous plsp1-1 (F) cotyledons. Plastoglobules are indicated with asterisks. (G and H) Plastid in 7-wk-old wild-type (G) and homozygous plsp1-1 (H) etiolated seedlings. White and black arrowheads indicate PLB and disorganized membrane structure, respectively. (E–H) Bars, 1 μm.

Mentions: We cloned a coding sequence of Plsp1 by RT-PCR from A. thaliana seedlings. Our initial attempts to express it and assay the activity of Plsp1 protein in E. coli were, however, unsuccessful. Thus, we took a genetic approach to test whether Plsp1 is involved in the maturation of Toc75. We identified a T-DNA mutagenized line of A. thaliana with an insertion in the fourth intron of PLSP1 and named it plsp1-1 (Fig. 2 A). Plants that were heterozygous for the insertion were apparently indistinguishable from wild type. All of their seeds were germinated on plates containing Murashige-Skoog (MS) media with 1% sucrose. About 25% of resultant seedlings were albino (Fig. 2 B) and died before they developed complete true leaves. We confirmed that they were homozygous for the insertion by genomic PCR (Fig. 2 C, lanes 4 and 5). Furthermore, PLSP1 transcript was not detectable in mutant plants (Fig. 2 D, lane 2). The seedling lethal phenotype of homozygous plsp1-1 plants was rescued by expressing a Plsp1 coding sequence with Cauliflower mosaic virus 35S promoter (Fig. 2, B–D). These data confirm that the phenotype of the mutant plants was caused by the disruption of PLSP1.


Complete maturation of the plastid protein translocation channel requires a type I signal peptidase.

Inoue K, Baldwin AJ, Shipman RL, Matsui K, Theg SM, Ohme-Takagi M - J. Cell Biol. (2005)

Disruption of PLSP1 results in a seedling lethal phenotype and reduction of the development of plastid internal membranes. (A) Structure of PLSP1. Gray boxes and solid lines indicate exons and introns, respectively. The position of the T-DNA insertion in plsp1-1 was determined experimentally and is indicated with a white arrowhead. Positions of primers used for genomic and RT-PCR are indicated with black arrows. Primers and inserted T-DNA indicated with arrows/arrowhead are not to scale. (B) Seedlings of wild-type (wt), plsp1-1, and plsp1-1;PLSP1 plants grown on MS media supplemented with 1% sucrose for 2 wk. Bar, 1 cm. (C) Genomic PCR of wild-type and mutant A. thaliana seedlings. E, I, and C indicate reactions specific to amplify endogenous PLSP1, the T-DNA insertion, and the transgene used to complement the mutation, respectively. Primers that were used are listed as follows: F2 and R1 for E, F2 and R3 for I, and primers from 35S promoter (forward) and nopaline synthase terminator (reverse) for C. (D) RT-PCR analyses of wild-type and mutant A. thaliana seedlings. Each reaction contained two sets of primers: one for PLSP1 cDNA (F1 and R2) that produces a 400-bp fragment and another for cDNA derived from 18S RNA that produces a 315-bp fragment. Images from different portions of the same gel are in separate boxes. (E and F) Plastids in 2-wk-old wild-type (E) and homozygous plsp1-1 (F) cotyledons. Plastoglobules are indicated with asterisks. (G and H) Plastid in 7-wk-old wild-type (G) and homozygous plsp1-1 (H) etiolated seedlings. White and black arrowheads indicate PLB and disorganized membrane structure, respectively. (E–H) Bars, 1 μm.
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fig2: Disruption of PLSP1 results in a seedling lethal phenotype and reduction of the development of plastid internal membranes. (A) Structure of PLSP1. Gray boxes and solid lines indicate exons and introns, respectively. The position of the T-DNA insertion in plsp1-1 was determined experimentally and is indicated with a white arrowhead. Positions of primers used for genomic and RT-PCR are indicated with black arrows. Primers and inserted T-DNA indicated with arrows/arrowhead are not to scale. (B) Seedlings of wild-type (wt), plsp1-1, and plsp1-1;PLSP1 plants grown on MS media supplemented with 1% sucrose for 2 wk. Bar, 1 cm. (C) Genomic PCR of wild-type and mutant A. thaliana seedlings. E, I, and C indicate reactions specific to amplify endogenous PLSP1, the T-DNA insertion, and the transgene used to complement the mutation, respectively. Primers that were used are listed as follows: F2 and R1 for E, F2 and R3 for I, and primers from 35S promoter (forward) and nopaline synthase terminator (reverse) for C. (D) RT-PCR analyses of wild-type and mutant A. thaliana seedlings. Each reaction contained two sets of primers: one for PLSP1 cDNA (F1 and R2) that produces a 400-bp fragment and another for cDNA derived from 18S RNA that produces a 315-bp fragment. Images from different portions of the same gel are in separate boxes. (E and F) Plastids in 2-wk-old wild-type (E) and homozygous plsp1-1 (F) cotyledons. Plastoglobules are indicated with asterisks. (G and H) Plastid in 7-wk-old wild-type (G) and homozygous plsp1-1 (H) etiolated seedlings. White and black arrowheads indicate PLB and disorganized membrane structure, respectively. (E–H) Bars, 1 μm.
Mentions: We cloned a coding sequence of Plsp1 by RT-PCR from A. thaliana seedlings. Our initial attempts to express it and assay the activity of Plsp1 protein in E. coli were, however, unsuccessful. Thus, we took a genetic approach to test whether Plsp1 is involved in the maturation of Toc75. We identified a T-DNA mutagenized line of A. thaliana with an insertion in the fourth intron of PLSP1 and named it plsp1-1 (Fig. 2 A). Plants that were heterozygous for the insertion were apparently indistinguishable from wild type. All of their seeds were germinated on plates containing Murashige-Skoog (MS) media with 1% sucrose. About 25% of resultant seedlings were albino (Fig. 2 B) and died before they developed complete true leaves. We confirmed that they were homozygous for the insertion by genomic PCR (Fig. 2 C, lanes 4 and 5). Furthermore, PLSP1 transcript was not detectable in mutant plants (Fig. 2 D, lane 2). The seedling lethal phenotype of homozygous plsp1-1 plants was rescued by expressing a Plsp1 coding sequence with Cauliflower mosaic virus 35S promoter (Fig. 2, B–D). These data confirm that the phenotype of the mutant plants was caused by the disruption of PLSP1.

Bottom Line: Next, we show that disruption of a gene encoding plastidic SPase I (Plsp1) resulted in the accumulation of immature forms of Toc75, severe reduction of plastid internal membrane development, and a seedling lethal phenotype.These phenotypes were rescued by the overexpression of Plsp1 complementary DNA.Plsp1 appeared to be targeted both to the envelope and to the thylakoidal membranes; thus, it may have multiple functions.

View Article: PubMed Central - PubMed

Affiliation: Department of Plant Sciences, College of Agricultural and Environmental Sciences, University of California, Davis, CA 95616, USA. kinoue@ucdavis.edu

ABSTRACT
The protein translocation channel at the plastid outer envelope membrane, Toc75, is essential for the viability of plants from the embryonic stage. It is encoded in the nucleus and is synthesized with a bipartite transit peptide that is cleaved during maturation. Despite its important function, the molecular mechanism and the biological significance of the full maturation of Toc75 remain unclear. In this study, we show that a type I signal peptidase (SPase I) is responsible for this process. First, we demonstrate that a bacterial SPase I converted Toc75 precursor to its mature form in vitro. Next, we show that disruption of a gene encoding plastidic SPase I (Plsp1) resulted in the accumulation of immature forms of Toc75, severe reduction of plastid internal membrane development, and a seedling lethal phenotype. These phenotypes were rescued by the overexpression of Plsp1 complementary DNA. Plsp1 appeared to be targeted both to the envelope and to the thylakoidal membranes; thus, it may have multiple functions.

Show MeSH