Identification of a Role for an Azide-Sensitive Factor in the Thylakoid Transport of the 17-Kilodalton Subunit of the Photosynthetic Oxygen-Evolving Complex

Abstract

We have examined the transport of the precursor of the 17-kD subunit of the photosynthetic O2-evolving complex (OE17) in intact chloroplasts in the presence of inhibitors that block two protein-translocation pathways in the thylakoid membrane. This precursor uses the transmembrane pH gradient-dependent pathway into the thylakoid lumen, and its transport across the thylakoid membrane is thought to be independent of ATP and the chloroplast SecA homolog, cpSecA. We unexpectedly found that azide, widely considered to be an inhibitor of cpSecA, had a profound effect on the targeting of the photosynthetic OE17 to the thylakoid lumen. By itself, azide caused a significant fraction of mature OE17 to accumulate in the stroma of intact chloroplasts. When added in conjunction with the protonophore nigericin, azide caused the maturation of a fraction of the stromal intermediate form of OE17, and this mature protein was found only in the stroma. Our data suggest that OE17 may use the sec-dependent pathway, especially when the transmembrane pH gradient-dependent pathway is inhibited. Under certain conditions, OE17 may be inserted across the thylakoid membrane far enough to allow removal of the transit peptide, but then may slip back out of the translocation machinery into the stromal compartment.

Figure 9

A, Reactions run in the absence of inhibitor; B, reactions run in the presence of azide; C, reactions run in the presence of nigericin; and D, reactions run in the presence of azide and nigericin. Lanes 1, Intact chloroplasts; lanes 2, isolated thylakoids; lanes 3, stromal fraction; lanes 4, mock protease-treated thylakoids; and lanes 5, protease-treated thylakoids. Intact chloroplasts were preincubated with nigericin (6 μm) and azide (30 mm) (or equivalent volumes of ethanol and water) for 10 min in the dark on ice. Import reactions with precursor OE23 and subsequent fractionations were performed as described in Figure 4, except that an aliquot of thylakoids was removed before the protease treatment. The standard (std) represents 20% of the precursor added to each reaction; the positions of the precursor (p), intermediate (i), and mature (m) proteins are noted.

Figure 1

Azide restores OE17 processing in the presence of uncouplers. Where indicated, isolated intact chloroplasts were preincubated with 30 mm azide, 15 μm CCCP, and 6 μm nigericin (or equivalent volumes of water or ethanol) for 10 min in the dark on ice. To start the reactions, the chloroplasts were added to the import reaction mixture (final concentrations of inhibitors were 10 mm azide, 5 μm CCCP, and 2 μm nigericin) and placed in light for 30 min. Reactions were terminated by centrifuging into PCA as described in Methods. The standard (std) represents 20% of the prOE17 added to each reaction; the positions of the precursor (p), intermediate (i), and mature (m) proteins are noted.

Figure 8

Intact chloroplasts (lanes 5–8) and washed thylakoids isolated from intact chloroplasts (lanes 1–4) were preincubated for 10 min in the dark with 6 μm nigericin, 30 mm azide, or equivalent volumes of ethanol and water. Reactions were initiated by the 3-fold dilution of chloroplasts or thylakoids into the import mixture and proceeded for 30 min in the light. Chloroplasts and thylakoids were reisolated by centrifugation into PCA through Wacker AR200 and a 50:50 (v/v) ratio of Wacker AR20 and AR200 silicon oils, respectively. The standard (std) represents 20% of the precursor added to each reaction; the positions of the precursor (p), intermediate (i), and mature (m) proteins are noted.

Figure 7

A, Stromal extract does not process prOE17 to its mature form. B, Thylakoid membranes are required for maturation. Lysis buffer or concentrated stromal extract collected from the lysis of chloroplasts at 6 mg chlorophyll/mL were preincubated with 6 μm nigericin (or ethanol) and 30 mm azide (or water) for 10 min on ice in the dark. These solutions were added to 1× IB containing radiolabeled prOE17, 5 mm MgCl₂, and 5 mm ATP, with no chloroplast membranes (A) or in the presence of thylakoid membranes (B). The samples were incubated in the light for 30 min and terminated by the addition of 2× sample buffer without further purification. The standard (std) represents 20% of the precursor added to each reaction; the positions of the precursor (p), intermediate (i), and mature (m) proteins are noted.

Figure 3

Import reactions were initiated by the addition of chloroplasts to 1× IB (final concentration in the reaction was 0.33 mg/mL chlorophyll) containing radiolabeled prOE17, 5 mm MgCl₂ and 5 mm ATP. A, Reactions incubated in the absence of both inhibitors; B, reactions incubated with azide alone; C, reactions incubated with nigericin alone; and D, reactions incubated with both inhibitors. Lanes 1, Intact chloroplasts; lanes 2, washed thylakoids (containing contaminating envelope membranes); lanes 3, supernatant fraction after envelope lysis of sample in lanes 2 containing stromal components and envelope membranes; and lanes 4, protease-treated thylakoids. The reactions were treated as in Figure 1. After this incubation, the samples were placed back in the light at 25°C for an additional 15 min. The chloroplast envelopes in lanes 2 and 4 were osmotically lysed in lysis buffer (see Methods) containing 2 μm nigericin, whereas the samples in lanes 1 were maintained in isotonic buffer (1× IB with 2 μm nigericin). Reactions were incubated on ice in the dark for 5 min. The osmoticum was then restored to samples in lanes 2 and 4 with the addition of 2× IB containing 2 μm nigericin (an equivalent volume of 1× IB with 2 μm nigericin was added to the samples in lanes 1). Samples in lanes 4 were protease treated with thermolysin (200 μg/mL with 5 mm CaCl₂); samples in lanes 1 and 2 received 5 mm CaCl₂ only. After inactivating thermolysin with the addition of 25 mm EDTA, membranes were pelleted and the supernatant collected from the samples in lanes 2. This supernatant was precipitated with 1.5 m PCA and loaded in lane 3. The standard (std) represents 20% of the precursor added to each reaction; the positions of the precursor (p) and mature (m) proteins are noted.

Figure 2

A, Increasing concentrations of nigericin do not abolish OE17 processing in the presence of azide. Intact chloroplasts were preincubated with 0, 6, 15, or 30 μm nigericin (always with the same volume of ethanol) in the dark for 10 min on ice in the presence or absence of 30 mm azide (or an equivalent volume of water). Chloroplasts were diluted 3-fold into the import mixture to start the assay, and reactions were allowed to continue for 30 min in the light. The import assay was terminated and the samples analyzed as described for Figure 1. The standard (std) represents 20% of the precursor added to each reaction; the positions of the precursor (p), intermediate (i), and mature (m) proteins are noted. B, Azide does not inactivate the uncoupling activity of nigericin. Isolated thylakoids were preincubated with 6 μm nigericin (or an equivalent volume of ethanol) and 30 mm azide (or an equivalent volume of water) for 10 min on ice in the dark. Thylakoids were then diluted 3-fold into import buffer containing 20 μm methyl viologen and 3 mm MgCl₂ to a concentration of 20 μg/mL. 9-Aminoacridine (5 μm) was added and the fluorescence was measured. Saturating red actinic light was turned on and off at 20 and 100 s; the shutter in front of the measuring beam was briefly shut at 75 s to determine if the sample chamber was light tight.

Figure 4

A, Reactions incubated in the absence of both inhibitors; B, reactions incubated with azide alone; C, reactions incubated with nigericin alone; and D, reactions incubated with both inhibitors. Lanes 1, Intact chloroplasts; lanes 2, stromal fractions; lanes 3, washed thylakoids; and lanes 4, protease-treated thylakoids. A 4× import reaction was initiated by the addition of 80 μg of chlorophyll and a 30-min import reaction was conducted in the light. At this time, one aliquot was removed and the sample was centrifuged through silicon oil into PCA; these intact chloroplasts were run in lanes 1. The remaining chloroplasts were pelleted for 30 s in a microfuge, the supernatant was removed, and the chloroplasts were resuspended in 0.5 volume of envelope lysis buffer containing 4 μg of aprotinin, 4 μg of leupeptin, and 1 mm PMSF. To distinguish between mOE17 bound to the lumenal face of the membrane and proteins that were fully translocated, we protease treated one-half of the thylakoids with thermolysin (lanes 4), whereas the other half was mock-protease treated (lanes 3). The thylakoid fraction also contained fragmented envelope membranes; thus, some prOE17 is seen in the samples that are not protease treated (lanes 3). The standard (std) represents 20% of the precursor added to each reaction; the positions of the precursor (p), intermediate (i), and mature (m) proteins are noted.

Figure 5

Intact chloroplasts were preincubated with OAA/DHAP (3 mm), azide (30 mm), nigericin (6 μm), or equivalent volumes of 1× IB, water, or ethanol, respectively, for 10 min on ice in the dark. Reactions were initiated by the 3-fold dilution of chloroplasts into the import mixture and continued for 30 min in the light. The assay was terminated and samples were analyzed as described in Figure 1. The standard (std) represents 20% of the precursor added to each reaction; the positions of the precursor (p), intermediate (i), and mature (m) proteins are noted.

Figure 6

Chloroplasts were preincubated with 1 mm PMSF (striped bars) or an equivalent volume of isopropanol (open bars) in the presence of nigericin (6 μm) and azide (30 mm) (or equivalent volumes of ethanol and water) for 10 min on ice in the dark. These chloroplasts were then diluted 3-fold into an import mixture containing 1 mm PMSF to initiate the protein-transport assay. Reactions proceeded for 30 min in the light and were terminated by repurifying the chloroplasts through silicon oil. Samples were separated by SDS-PAGE, visualized by fluorography, and the fluorographs were quantitated by densitometry. %IOD represents mOE17 as a fraction of the total amount of radiolabeled protein found in the sample. The data shown represent the average from three experiments.