Evaluating the Functional Pore Size of Chloroplast TOC and TIC Protein Translocons: Import of Folded Proteins

Abstract

The degree of residual structure retained by proteins while passing through biological membranes is a fundamental mechanistic question of protein translocation. Proteins are generally thought to be unfolded while transported through canonical proteinaceous translocons, including the translocons of the outer and inner chloroplast envelope membranes (TOC and TIC). Here, we readdressed the issue and found that the TOC/TIC translocons accommodated the tightly folded dihydrofolate reductase (DHFR) protein in complex with its stabilizing ligand, methotrexate (MTX). We employed a fluorescein-conjugated methotrexate (FMTX), which has slow membrane transport rates relative to unconjugated MTX, to show that the rate of ligand accumulation inside chloroplasts is faster when bound to DHFR that is actively being imported. Stromal accumulation of FMTX is ATP dependent when DHFR is actively being imported but is otherwise ATP independent, again indicating DHFR/FMTX complex import. Furthermore, the TOC/TIC pore size was probed with fixed-diameter particles and found to be greater than 25.6 Å, large enough to support folded DHFR import and also larger than mitochondrial and bacterial protein translocons that have a requirement for protein unfolding. This unique pore size and the ability to import folded proteins have critical implications regarding the structure and mechanism of the TOC/TIC translocons.

Figure 1.

Structural Models for DHFR/FMTX Import. (A) Model 1: The DHFR/FMTX complex is unfolded at the chloroplast surface; both components are imported independently across the envelope membranes (OM and IM) and reassociate in the stroma. Model 2: The DHFR/FMTX complex remains intact during import across the envelope membranes. (B) The rate of free FMTX import (R_{FMTX, f}) in the absence of DHFR import is compared with the theoretical rates of FMTX import in Models 1 and 2 (R_FMTX1 and R_FMTX2) to establish a basis for differentiating the two models.

Figure 2.

Fluorescence Enhancement of FMTX upon Binding tp22DHFR. tp22DHFR (40 nM) was added to 80 nM FMTX in import buffer followed by addition of excess MTX (800 nM) at indicated time points.

Figure 3.

FMTX Import into Chloroplasts Is Dependent upon Concurrent DHFR Import. (A) Schematic of FMTX import experiment. tp22DHFR (930 nM) was imported into chloroplasts in 300-μL reactions with or without 8 μM FMTX as shown (initial import). Reactions were stopped with cold import buffer. Chloroplasts were washed, resuspended in 400 μg/mL thermolysin, incubated 1 h on ice, and quenched with 12.5 mM EDTA. Chloroplasts were resuspended in import buffer with ATP and with or without 8 μM FMTX as indicated and were again exposed to light for 20 min (mock import). Chloroplasts were reisolated on 40% Percoll cushions, washed with import buffer, and assayed for fluorescence in 3 mL of import buffer. (B) Fluorescence of chloroplasts derived from DHFR and FMTX co-import (traces 1 and 2) or independent import treatments (traces 3 and 4). Trace 5 represents background chloroplast fluorescence derived from chloroplasts treated as shown in (A), except without any addition of protein or FMTX. Excess MTX (17 μM, traces 2 and 4) or DMSO (traces 1, 3, and 5) was added at t = 2 min, and 0.05% Triton X-100 (TX) was added to all traces at t = 4 min. All traces represent averages of three independent experiments. (C) RSSUFHC precursor protein labeled with fluorescein maleimide was imported into chloroplasts, which were thermolysin treated, reisolated, and assayed for fluorescence (+F) along with a mock-import control (−F). (D) DMSO-added traces from (B) were subtracted from MTX-added traces (traces 2−1 and 4−3) to show the MTX replacement reaction kinetics. (E) Background fluorescence (trace 5) was subtracted from traces 1 through 4. Replicates were offset adjusted to the same initial fluorescence. tp22DHFR/FMTX complex import was determined as the change in fluorescence due to debinding of FMTX from DHFR (base fluorescence at t = 21 min in MTX added trace subtracted from peak fluorescence at t = 5 min in the DMSO added trace). Inset: Fluorescence change (ΔF) was calibrated by adding DHFR lacking tp22 fusion in 14 pmol increments to a saturating 170 nM FMTX solution in import buffer containing chloroplasts (17 μg/mL chlorophyll). (F) The true tp22DHFR/FMTX peak at t = 4 min was determined by extrapolating linearized DMSO-added traces (1 and 3) back to t = 4 min. Decay curves were linearized by taking the natural log of normalized data. Sample extrapolation is shown. (G) tp22DHFR import was determined by α-FLAG blotting, shown along with Rubisco small subunit (RSSU) as a loading control. (H) tp22DHFR protein import and tp22DHFR/FMTX complex import (nonextrapolated and extrapolated) were quantified from immunoblots and fluorescence data, respectively. Error bars indicate se (n = 3). CBB, Coomassie Brilliant Blue; pr, tp22DHFR precursor; m, mature tp22DHFR.

Figure 4.

Kinetics of DHFR/FMTX Complex and FMTX Import. Chloroplasts were treated with tp22DHFR and FMTX as in Figure 3A, except that import reactions were stopped at various time points. DHFR/FMTX complex import kinetics were determined by stopping the initial import reaction at the indicated time points and stopping the second mock import reaction after 12 min (co-import, green squares). FMTX import kinetics were determined by stopping the initial import reaction (with tp22DHFR) after 12 min and stopping the second mock import reaction (with FMTX) at the indicated time points (independent import, gray circles). Samples were assayed for stromal DHFR/FMTX as in Figure 3. The 12-min co-import time points were normalized to 1. Error bars indicate sd (n = 3). Lines show data fitted with a single rising exponential, both approaching the same maximum. The DHFR/FMTX co-import rate constant was 2.9-fold greater than the independent import rate constant.

Figure 5.

Concurrent Inhibition of DHFR and FMTX Import into Chloroplasts. (A) tp22DHFR (1.3 μM) was preimported into chloroplasts for 8 min at room temperature under illumination (hν) (no exogenous ATP was added), after which reactions were stopped on ice. Stromal ATP was depleted with 10 mM glycerate (Glyc.), 5 μM nigericin (Nig.), and 5 μM valinomycin (Val.) on ice for 5 min (ATP depletion). Control reactions (no ATP depletion) were mock treated with appropriate volumes of solvents (import buffer, ethanol, and DMSO). FMTX (15 μM) was added to the cold reactions at this point to allow binding to external DHFR. ATP-depleted and control reactions without ATP depletion were incubated at room temperature under illumination for an additional 6 min. Import reactions were stopped with cold import buffer. Chloroplasts were thermolysin treated and reisolated through Percoll as in Figure 3. (B) Chloroplasts were treated essentially as in the Figure 3A independent import treatment, except that the second mock import reaction was conducted with 10 mM glycerate, 5 μM nigericin, and 5 μM valinomycin (ATP depletion) or solvent controls (no ATP depletion). Also, the initial import reaction with tp22DHFR and subsequent mock import reaction with FMTX were conducted for 16 and 12 min, respectively, at room temperature under illumination with no exogenously added ATP. All samples in (A) and (B) were assayed for stromal DHFR/FMTX by extrapolated fluorescence or immunoblotted for stromal DHFR as in Figure 3. Representative blots and averaged fluorescence traces corrected for background chloroplast fluorescence are shown. Quantifications are shown with se (n = 3). Significant differences were determined by t tests (*P < 0.05). Quantifications with and without stromal ATP depletion in (A) were subtracted to show change in import (gray bars) for DHFR (bar 2−1) and FMTX (bar 4−3).

Figure 6.

The TOC/TIC Pore Size Is Greater Than 20 Å. Undecagold (UG)-labeled (A) or fluorescein (F)-labeled (B) RSSUFHC was localized to the chloroplast stroma. Import reactions were conducted for 20 min and stopped in cold import buffer (Imp). For protease treatment, chloroplasts were further resuspended in import buffer containing 400 μg/mL thermolysin (Therm), incubated 30 min on ice, and quenched with 12.5 mM EDTA. All samples were reisolated on 40% Percoll cushions and washed with import buffer. After reisolation, some non-protease-treated samples were separated into membrane (M) and soluble (S) fractions by lysis in 2 mM EDTA for 10 min on ice, mixing with 200 mM NaCl, and pelleting membranes at 16,000g. Soluble fractions were precipitated in 15% TCA on ice and washed with cold acetone. Samples were analyzed by SDS-PAGE and α-FLAG blotting along with percent input standards. Mature (M) labeled protein was quantified and, as an internal control, the unlabeled mature protein in the same lanes as the labeled protein was also quantified. Error bars indicate se (n = 3). Labeled protein Imp treatments were normalized to 1. pr, RSSUFHC precursor; m, mature RSSUFHC.

Figure 7.

The TOC/TIC Pore Size Is Greater Than 25.6 Å. Biotinylated RSSUHC (RSSUHC-Bt, 8.3 μg/mL in import reaction) was incubated with 17.5- to 70-fold molar excess mSA in import buffer prior to import reactions. Thermolysin treatments were conducted as in Figure 6. Stromal localization was determined as in Figure 6, except that the membrane/stroma fractionation was done on thermolysin-treated chloroplasts. For trypsin (Tryp) treatments, postimport chloroplasts were resuspended in import buffer containing 240 μg/mL trypsin, incubated 30 min at 25°C, resuspended again in 1 mg/mL soybean trypsin inhibitor, reisolated on 40% Percoll, and washed with import buffer. Additionally, protease treatments were conducted in the presence of 1% Triton X-100 (TX). RSSUHC-Bt and mSA were detected on α-biotin and α-FLAG blots, respectively, along with standards that represent a molar percentage of RSSUHC-Bt input. Error bars indicate se (n = 3). mSA-labeled mature RSSUHC or mSA from the Therm treatment were normalized to 1. pr, RSSUHC-Bt precursor; m, mature RSSUHC-Bt. Asterisk indicates nonspecific chloroplast protein.

Figure 8.

Import Efficiencies of Fixed-Diameter Probes. (A) and (B) Eight-minute import reactions of fluorescein (F)-labeled and Undecagold (UG)-labeled RSSUFHC (A) and mSA-labeled RSSUHC-Bt (B) are shown. RSSUHC-Bt (8.3 μg/mL in import reaction) was incubated with a 70-fold molar excess of mSA in import buffer prior to import. All reactions were stopped after 8 min with cold 400 μg/mL thermolysin and incubated 1 h on ice. Samples were subsequently treated as in Figures 6 and 7. Mature RSSU (mRSSUHC-Bt) and mSA were quantified to illustrate their equimolar import ratio. (C) Import efficiencies of all three labeled proteins (defined as the labeled:unlabeled mature protein ratio) were plotted as a function of probe size (black circles). The internal control unlabeled protein was used for fluorescein and Undecagold quantifications. Error bars (sd) represent multiple replicates from two independent experiments. For comparison, tp22DHFR/FMTX import efficiency was plotted as the ratio of tp22DHFR import in the presence and absence of FMTX derived from Figure 3H (green circle, error bar = sd, n = 3). Protein dimensions were plotted as minor axis diameters with error bars representing sd away from a perfectly circular cross section in the minor axis plane. Mitoplast TIM complex import rates for fluorescein (13 Å), Undecagold (20 Å), and Nanogold (26 Å) size probes were taken from Schwartz and Matouschek (1999). The Undecagold import efficiency was plotted as the labeled:unlabeled ratio of preprotein extent of import after 4 min since the authors noted that the initial import rates overestimate the import efficiency. Bacterial SecYEG transport rates for tetraarylmethane derivative size probes (TAMs) were replotted from Bonardi et al. (2011) with the unlabeled substrate transport rate normalized to 1. Internally cross-linked BPTI translocation efficiency through TOC/TIC, TIM, and SecYEG complexes (cyan circle/square/triangle) and DHFR/MTX translocation efficiency through TIM and SecYEG complexes (green square/triangle) also derive from previous publications (Eilers and Schatz, 1986; Schiebel et al., 1991; Jascur et al., 1992; Arkowitz et al., 1993; Clark and Theg, 1997). Asterisk indicates nonspecific chloroplast protein.