Chloroplast import motor subunits FtsHi1 and FtsHi2 are located on opposite sides of the inner envelope membrane

Abstract

Protein import into chloroplasts is powered by ATP hydrolysis in the stroma. Establishing the identity and functional mechanism of the stromal ATPase motor that drives import is critical for understanding chloroplast biogenesis. Recently, a complex consisting of Ycf2, FtsHi1, FtsHi2, FtsHi4, FtsHi5, FtsH12, and malate dehydrogenase was shown to be important for chloroplast protein import, and it has been proposed to act as the motor driving protein translocation across the chloroplast envelope into the stroma. To gain further mechanistic understanding of how the motor functions, we performed membrane association and topology analyses on two of its subunits, FtsHi1 and FtsHi2. We isolated cDNA clones encoding FtsHi1 and FtsHi2 preproteins to perform in vitro import experiments in order to determine the exact size of each mature protein. We also generated antibodies against the C-termini of the proteins, i.e., where their ATPase domains reside. Protease treatments and alkaline and high-salt extractions of chloroplasts with imported and endogenous proteins revealed that FtsHi1 is an integral membrane protein with its C-terminal portion located in the intermembrane space of the envelope, not the stroma, whereas FtsHi2 is a soluble protein in the stroma. We further complemented an FtsHi1-knockout mutant with a C-terminally tagged FtsHi1 and obtained identical results for topological analyses. Our data indicate that the model of a single membrane-anchored pulling motor at the stromal side of the inner membrane needs to be revised and suggest that the Ycf2-FtsHi complex may have additional functions.

Keywords: chloroplast; protein import; protein topology.

Fig. 1.

Chloroplast import of prFtsHi1 and prFtsHi2. (A) The proposed model for the FtsHi import motor. The one dark green and five light green units indicate Ycf2 and the five FtsH/FtsHi subunits, respectively. MDH, malate dehydrogenase. (B) Schematic representation of the Arabidopsis prFtsHi1 and prFtsHi2 polypeptides. The predicted transit peptides, transmembrane spans, and the Walker A motifs for nucleotide binding are colored in green, blue, and purple, respectively. The regions used for antibody production are colored red and the sequences of the peptide antigens are shown. The number next to each predicted transmembrane span is the consensus score from AramTmCon. (C and D) [³⁵S-Met]-prFtsHi1 (C) and prFtsHi2 (D) were imported into isolated pea chloroplasts. Preproteins before import and chloroplasts after import were treated with 100 μg/mL thermolysin or 200 μg/mL trypsin or chymotrypsin and analyzed by SDS-PAGE and fluorography. The chloroplast samples were also analyzed by immunoblotting to reveal amounts of Tic110 and Toc75. (E) Topology and membrane association of FtsHi1 and FtsHi2. The exact number of FtsHi1 transmembrane spans is not known, so its transmembrane region is represented by a rectangle and the location of its N terminus is speculative. Purple ovals marked with ATP indicate the Walker A motifs.

Fig. 2.

The C-terminal portion of FtsHi1 is located in the intermembrane space. (A) Isolated Arabidopsis chloroplasts were treated with 200 μg/mL trypsin or 200 μg/mL trypsin plus 200 μg/mL chymotrypsin. Blue arrow, FtsHi1; asterisks, nonspecific cross-reacting proteins. (B) Same as in A except the chloroplasts were treated with 100 μg/mL thermolysin. (C) Quantification of experiments shown in A and B. Signals for FtsHi1 and FtsHi2 were quantified and normalized to Tic110 signal of the same sample for the trypsin and chymotrypsin treatments and normalized to Toc75 signal of the same sample for the thermolysin treatments. Ratio to samples not treated with proteases in the same experiment was then calculated. Value = mean ± SD, n = 3. (D) Isolated Arabidopsis chloroplasts were treated with 250 μg/mL trypsin and with or without 1% Triton X-100. (A, B, and D) After protease digestion, the chloroplasts were reisolated and analyzed by SDS-PAGE, followed by immunoblotting with antibodies against the proteins indicated. An equal amount of proteins was loaded in all lanes of the same panel.

Fig. 3.

FtsHi2 is a soluble protein in the stroma. (A) Isolated Arabidopsis chloroplasts were lysed using a hypotonic, alkaline or high-salt extraction buffer. After the treatment, lysed chloroplasts were separated into supernatant (S) and pellet (P) fractions by ultracentrifugation. Samples were then analyzed by SDS-PAGE, followed by immunoblotting with antibodies against the proteins indicated at right. Fifty μg of protein was loaded in each pellet lane. Each supernatant lane contained the same number of chloroplasts as its corresponding pellet lane from the same treatment. (B) Isolated Arabidopsis chloroplasts were resuspended in a hypertonic buffer, lysed by freezing and thawing and fractionated by sucrose density gradient. An equal amount of proteins was loaded in the four subchloroplast fractions of the same panel.

Fig. 4.

Complementation of ftshi1-2 with FtsHi1cDNA-Myc. (A) Schematic representation of FtsHi1 genomic DNA and the T-DNA insertion position in the ftshi1-2 allele. Numbers below the genomic DNA indicate the nucleotide number. Filled boxes, exons; open boxes, introns; LB, left border of the T-DNA. The ftshi1-2 mutant has multiple T-DNA inserted in the same position with left borders at both ends (21). (B) Siliques from an ftshi1-2 homozygous mutant complemented with the FtsHi1cDNA-Myc transgene (Left) and a plant heterozygous for the ftshi1-2 T-DNA insertion (+/ftshi1-2, right). Bar = 1 mm. (C) Genotyping of a wild-type (WT) and an ftshi1-2 homozygous mutant complemented with the FtsHi1cDNA-Myc transgene. Total genomic DNA was isolated and amplified by PCR with primers indicated below the lanes. Positions of primers are marked in A. The LB3 primer of the T-DNA left border (LB) was used for the LB position. (D) Twenty-day-old seedlings of the wild type and ftshi1-2 homozygous mutant complemented with FtsHi1cDNA-Myc. Bar = 1 cm. (E) Chloroplasts isolated from ftshi1-2 homozygous mutant plants complemented with FtsHi1cDNA-Myc were treated with 100 μg/mL thermolysin or 200 μg/mL trypsin plus 200 μg/mL chymotrypsin, analyzed by SDS-PAGE, and then subjected to immunoblotting with antibodies against the proteins indicated. Asterisks, proteins cross-reacting with the anti-FtsHi1 antiserum.