©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
PSII-T, a New Nuclear Encoded Lumenal Protein from Photosystem II
TARGETING AND PROCESSING IN ISOLATED CHLOROPLASTS (*)

Aliki Kapazoglou (§) , Francis Sagliocco (¶) , Leon Dure III (**)

From the (1) Department of Biochemistry and Molecular Biology, University of Georgia, Athens, Georgia 30602

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

An intronless nuclear gene, psbT, isolated from cotton, encodes a putative 11-kDa protein (PSII-T) highly homologous in its C terminus to the N terminus of the partially sequenced PSII-T protein from spinach photosystem II. Analysis of the deduced amino acid sequence of cotton PSII-T revealed the presence of potential chloroplast stroma and thylakoid targeting domains and a putative mature PSII protein of 3.0 kDa, composed of only 28 amino acid residues. The cotton PSII-T 11-kDa precursor was synthesized in vitro in a wheat germ extract translation system, and the translation product was used in assays for protein imports into isolated pea chloroplasts. It was shown that the cotton PSII-T precursor was imported into the chloroplasts, processed to a mature form of approximately 3.0 kDa, agreeing with the predicted size from amino acid sequence analysis, and localized to the lumenal side of the thylakoid membrane, thus defining a new nuclear encoded lumenal protein and the smallest polypeptide of PSII reported to date. Processing of the PSII-T precursor occurred in two steps and involved the formation of a stromal intermediate of approximately 7.5 kDa, as predicted from primary structure analysis.


INTRODUCTION

In oxygen-evolving photosynthetic organisms, photosystem II (PSII)() is composed of three major complexes: the light-harvesting antennae, the reaction center core, and the oxygen-evolving complex (1) . A number of intermediate molecular weight polypeptides (>10 kDa) composing these complexes have been extensively studied and include the light-harvesting chlorophyll a/b binding proteins, the D1 and D2 proteins of the reaction core, and the oxygen-evolving (OE) complex polypeptides OE33, OE23, and OE17. Improvements in the resolution of polypeptides by SDS-PAGE techniques in the past few years has enabled the isolation of a number of low molecular weight proteins from PSII, mostly of unknown function and ranging in size from 3.6-10 kDa (2, 3, 4, 5) .

Many of these low mass PSII polypeptides are encoded in the nuclear genome in plants and algae, as judged by their absence in the four sequenced chloroplast genomes (1) . Nuclear encoded chloroplastic proteins are synthesized as higher molecular weight precursors with N-terminal extensions (chloroplast transit peptides), which direct their import across the chloroplast envelope into the stroma and are cleaved by a stromal processing protease (6) . Thylakoid membrane proteins like the light-harvesting chlorophyll a/b binding proteins are processed by the stromal protease to their mature form and are directed to the thylakoid membrane by targeting signals contained in the mature protein. Alternatively, thylakoid lumenal proteins such as the oxygen-evolving complex polypeptides, OE33, OE23, and OE17, contain bipartite presequences composed of the stroma targeting domains and thylakoid targeting domains, which direct them across the thylakoid membrane into the thylakoid lumen (7) . The stroma targeting domain is cleaved by the stromal protease, and the resulting intermediate is translocated into the thylakoid lumen where a thylakoid processing protease cleaves the thylakoid transfer domain resulting in the mature protein (7, 8, 9, 10) .

Stroma targeting peptides vary in size from 30 to 100 amino acids, and comparison of a large number of chloroplast precursors has defined the sequence VXAA as a loose consensus motif for stromal processing, with cleavage occurring between the two alanine residues (12) . The thylakoid transfer domain, on the other hand, contains a region very similar to the signal peptide of secreted proteins in prokaryotes and eukaryotes and contains a strongly conserved motif AXA preceding the processing site (11) .

One of the nuclear encoded low molecular weight PSII proteins, the ``5-kDa polypeptide,'' was first isolated from spinach thylakoids and purified to homogeneity by Ljungberg et al.(2) and found to be an extrinsic PSII membrane protein. An analog from wheat has also been isolated; the partial N-terminal amino acid sequence for both polypeptides has been determined for the first 29 and 10 amino acids, respectively (4) ; and the protein species has been named PSII-T and their genes psbT(1) .

We report here the isolation and characterization of a nuclear gene from cotton encoding a putative 11-kDa protein, highly homologous in its C-terminal region to the 5-kDa PSII-T proteins from spinach and wheat and containing potential stroma and thylakoid targeting domains. It is further demonstrated that the PSII-T precursor is imported into isolated pea chloroplasts, where it is localized to the lumenal side of the thylakoid membrane with a final size of approximately 3 kDa. Maturation of the precursor polypeptide to its final form occurs in two steps and involves the formation of a stromal intermediate.


MATERIALS AND METHODS

Restriction endonucleases, the Klenow fragment of polymerase I, and T4 DNA ligase were purchased from Boehringer Mannheim, and T3 RNA polymerase was purchased from Stratagene. Wheat germ extract was obtained from Promega Corp. Radiolabeled [S]methionine (>1000 Ci/mmol) and C-radiolabeled low molecular weight protein standards were from Amersham Corp., and thermolysin was from Sigma.

Gene Isolation, Sequencing, and Amino Acid Sequence Analysis

Construction of cDNA and genomic libraries from cotton (Gossypium hirsutum cv. Coker 201) have been described previously (13) . psbT cDNA was obtained from a light-induced cDNA library and used to screen a cotton phage 2001 genomic library, from which a genomic clone was isolated. The genomic clone was mapped, sequenced by dideoxy DNA sequencing, and analyzed for open reading frames. The deduced amino acid sequences were examined for sequence homologies with known proteins in the PIR and SWISS-PROT data banks using the Intelligenetics Suite software system.

Construction of in Vitro Transcription Plasmid

The 1933 nucleotide psbT genomic clone, which was found not to contain introns, was digested with AvaII and NdeI, and the resulting 613-nucleotide fragment (delineated with arrows in Fig. 1) was blunt ended with the Klenow fragment of polymerase I. The blunt ended fragment was then ligated to plasmid pBluescript II KS+ (Stratagene) at the EcoRV site.


Figure 1: Nucleotide sequence of the cotton psbT gene and deduced amino acid sequence of the encoded protein. The partial cDNA sequence is underlined, and differences from the gene sequence are shown in lowercaseletters. Putative CAAT box, TATA box, and polyadenylation signal AATAAT are shaded. GATA motifs, found in other plant light-regulated genes, are also shaded. Two arrows, in the 5`-untranslated region and 3`-untranslated region, indicate the AvaII and NdeII sites, respectively, which were used for the subcloning of the in vitro transcription template (see ``Materials and Methods''). The nucleotide and amino acid sequence have been submitted to GenBank and PIR, respectively, under the accession number X54092.



In Vitro Transcriptions and Translations

Recombinant plasmid carrying the 613-nucleotide fragment was tested for insert orientation by restriction analysis. The plasmid was linearized downstream of the inserted fragment with NotI, and the linearized template was transcribed in vitro using T3 RNA polymerase. The psbT RNA template was isolated and translated in vitro in the wheat germ system in the presence of [S]methionine, using Promega's technical manual for in vitro translation. Translations were analyzed by Tricine-SDS-PAGE (14) and fluorography.

Preparation of Chloroplasts and Thylakoids

Intact chloroplasts were isolated from 9-10-day-old pea seedlings (Pisum sativum L. cv. Laxton's Progress 9) as described by Cline (15) . Chloroplasts were resuspended in import buffer (IB) (50 mM Hepes/KOH, pH 8.0; 0.33 M sorbitol) and kept on ice until used. Chloroplast lysates were prepared by osmotic lysis at 0 °C in 10 mM Hepes/KOH, pH 8.0, containing 10 mM MgCl. Thylakoids were separated from the soluble chloroplast fraction by centrifuging lysates at 3200 g for 8 min at 4 °C and were washed twice with import buffer containing 10 mM MgCl.

Chloroplast Protein Import Assays and Chloroplast Fractionation

Import of radiolabeled PSII-T precursor into isolated chloroplasts was conducted in an illuminated water bath at 25 °C, for 15 min (15) . Import reactions in the light were conducted in a total volume of 600 µl. Reactions were initiated by adding 100 µl of radiolabeled precursor, diluted 4 with 30 mM nonradioactive methionine, to 400 µl of chloroplasts, corresponding to 200 µg of chlorophyll, and 100 µl of 60 mM Mg-ATP, pH 8.0. Import assays were stopped by transfer of the tubes to ice. Intact chloroplasts were reisolated from 150 µl of the import reaction mixture by centrifugation on 35% Percoll at 4 °C, 3200 g for 8 min. The chloroplasts were washed with 1 IB, pelleted again at 1000 g for 5 min, resuspended in 50 µl of 20 mM EDTA and 50 µl 2 SDS-PAGE sample buffer, and frozen at -20 °C.

From the remaining 450 µl of the import reaction mixture, chloroplasts were pelleted at 1000 g, resuspended in 0.5 ml of IB, and treated with 25 µl of 2 mg/ml thermolysin for 40 min on ice. The protease was inactivated by adding 250 µl of 50 mM EDTA in IB. Intact chloroplasts were reisolated by centrifugation on 35% Percoll containing 5 mM EDTA, washed with 5 mM EDTA in IB, and pelleted as above. Chloroplasts were lysed in 75 µl of 10 mM Hepes/KOH, pH 8.0, 10 mM MgCl, followed by addition of an equal volume of 2 import buffer. One-fourth of the lysate was frozen and represents protease-protected proteins imported into the chloroplasts (L+). The rest was fractionated into a soluble fraction (S) and membranes by centrifugation at 3200 g for 8 min. The membrane fraction was resuspended in 750 µl of IB, divided into three aliquots, and pelleted by centrifugation. One aliquot was resuspended in 37 µl of 20 mM EDTA and frozen (M-). The other two aliquots were treated with 30 µl of thermolysin in IB (0.33 mg/ml final concentration) in the presence (M + TX) and absence (M+) of Triton X-100 (1% final concentration). Thermolysin was inactivated with 7 µl of 250 mM EDTA in import buffer (50 mM final concentration). Membranes were solubilized by adding an equal volume of 2 SDS-PAGE sample buffer (14) and were immediately boiled to prevent degradation by residual protease activity.

Import assays in the dark were conducted in a 300-µl total volume at 25 °C in tubes protected from light by aluminum foil. Chloroplasts were separated into two aliquots and incubated with (D+) and without (D-) thermolysin as described above. Following protease inactivation with EDTA, unbroken chloroplasts were reisolated as before and washed with 5 mM EDTA in IB, pelleted at 1000 g for 10 min, and resuspended in 50 µl of 20 mM EDTA and 50 µl of 2 SDS sample buffer.

Protease Sensitivity of PSII-T Precursor

20 µl of radiolabeled precursor diluted 5 with 30 mM methionine was treated with 3 µl of 2 mg/ml thermolysin for 40 min at 4 °C, and the protease was inactivated with 3 µl of 250 mM EDTA in IB. An equal volume of 2 SDS sample buffer was added, and the tube was placed in boiling water for 5 min. To insure that protease sensitivity did not result from SDS denaturation prior to inactivation of protease by SDS/heating, the reaction was repeated with thermolysin and EDTA added together just prior to heating.

Time Course Import Assays

For time course import assays, two reactions were set up of total volume 4.8 ml, each containing 3.2 ml of chloroplasts (0.5 mg/ml), 800 µl of 60 mM ATP, and 800 µl of radiolabeled PSII-T precursor diluted 4 with nonradioactive 30 mM methionine. Chloroplasts were prewarmed for 5 min at 25 °C in an illuminated water bath prior to the addition of precursor. The reactions were terminated at 0, 0.5, 1.0, 2.0, 3.0, 5.0, and 10.0 min by transferring 600-µl aliquots to tubes containing 10 µl of 0.2 M HgCl on ice for 5 min. The two sets were treated with and without protease, as described above, and the final pellets were resuspended in 45 and 60 µl of 20 mM EDTA, respectively.

Analysis of Samples

All samples were visualized by Tricine-SDS-PAGE (14) and fluorography. Aliquots of each assay were precipitated with cold (-20 °C) acetone (95% final concentration), and centrifuged at 10,000 g for 10 min. The pellets were dried and solubilized in SDS-sample buffer, and aliquots were subjected to electrophoresis and fluorography as described in the figure legends. For time course assays, chlorophyll concentration was determined following acetone precipitation (16) , and equal amounts of protein were electrophoresed for each time point by adjusting the volume of sample buffer used to resuspend each pellet. Precipitation with acetone and use of the Tricine-SDS-PAGE system was necessary to resolve mature PSII-T, due to its small size and its co-migration with chlorophyll-SDS micelles in conventional SDS-PAGE.

RESULTS

Isolation and Characterization of the Cotton psbTGene

The nucleotide sequence of the 1933-nucleotide psbT genomic clone and the derived amino acid sequence are shown in Fig. 1 . The gene includes a putative coding region of 315 nucleotides containing no introns that would translate to a protein of 105 amino acids with molecular mass of 11 kDa. The cDNA sequence is underlined, and only three differences with the genomic sequence were found, all in the 3`-untranslated region. Potential regulatory elements in the 5` upstream and 3` downstream untranslated regions are indicated in Fig. 1. The most likely functional CAAT and TATA sequences are at positions -213 and -114, relative to the putative translation initiation site, respectively. Between these boxes, there are two elements, AAGATAATA (position -177) and CTGATAAGA (position -127) that strongly resemble a conserved GATA motif, AATGATAAGG, that has been found in this region in many light-regulated genes (17) . An AT-rich box found in several other plant genes (17) is also present. A putative AATAAT polyadenylation signal is indicated.

Analysis of Protein Sequence

Homology between the Cotton Putative PSII-T Protein and the Spinach PSII 5-kDa Protein

The deduced amino acid sequence of psbT was examined for statistically significant homologies with other proteins, and homology was found between the C terminus of cotton PSII-T and the partially sequenced (29-amino acid) N terminus of the spinach 5-kDa protein from PSII (4) as shown in Fig. 2 . Between the region spanning the last 28 C-terminal amino acids of cotton PSII-T and the N terminus of the spinach sequence there is at least 70% identity and two conservative replacements. It is likely that the two unidentified residues (X) in the spinach sequence are cysteine residues, as in the cotton deduced sequence, that are not preserved during protein sequencing. The 5-kDa value of the spinach protein may be an overestimation due to anomalous behavior of small proteins on SDS-PAGE, caused in part by the co-migration of lipids in thylakoid membrane protein extracts (5) .


Figure 2: Amino acid sequence comparison between the putative PSII-T cotton protein and the PSII 5-kDa protein from spinach. The twotoplines represent the cotton PSII-T precursor, and the bottomline represents the N-terminal sequence of the mature spinach protein. X, undetermined residue in that sequence; ?, the possible unsequenced C-terminal region of the protein. The region of homology is presented in boldfacetype; residue identity and conservative replacements are shown by straight and dottedlines, respectively. The putative stroma and thylakoidal processing sites of PSII-T precursor are marked by arrows1 and 2, respectively. The corresponding putative recognition sites VVANAA and ATA are shown with underlines. The potential thylakoid transfer domain is indicated by a singleunderline.



Targeting Domains

Examination of the 75-amino acid presequence segment revealed a potential stroma targeting domain and a thylakoid targeting domain (Fig. 2). The first 24-amino acid region of PSII-T contains features of stroma targeting domains (11) in that it has a high content of serine and threonine residues (12.5% Ser, 21.8% Thr), contains no acidic amino acid, and is composed of an uncharged N-terminal segment followed by a region containing four arginine residues (Fig. 2).

A stromal cleavage recognition site resembling the loose consensus VXAA (12) , could be presumed to be the VVANAA sequence with hypothetical cleavage site between the two Ala residues (Fig. 2, double underline). This would result in a putative stroma targeting domain of 32 amino acids with a molecular mass of 3.57 kDa and a putative PSII-T intermediate form of 73 amino acids with a molecular mass of 7.5 kDa. Within this putative intermediate resides a sequence strongly resembling the thylakoid transfer domains of chloroplast lumenal proteins (Fig. 2, singleunderline). It contains a short N-terminal segment of two arginines and a glutamate, followed by a 10-amino-acid hydrophobic segment, and a C terminus of 9 residues with the putative thylakoid processing site ATA (11) . Cleavage occurring after the ATA would yield a putative thylakoid targeting domain of 45 amino acids with molecular weight of 4.43 kDa and a putative mature protein of 28 amino acids with molecular weight of 3.0 kDa.

In order to determine whether the PSII-T precursor is imported into the chloroplast and localized to the thylakoid lumen, as suggested by targeting elements in the putative transit peptide, in vitro protein import of PSII-T into intact chloroplasts was investigated using the in vitro translation product synthesized from RNA, produced in turn from the T3 polymerase transcription of the recombinant plasmid carrying the 613-nucleotide insert.

In Vitro Import of Radiolabeled PSII-T Precursor into Isolated Pea Chloroplasts

Import assays were conducted in the light and in the presence of exogenously supplied ATP. Chloroplasts were then treated with thermolysin, to remove precursors bound to the envelope. Control assays were conducted in the dark without exogenously supplied ATP to verify ATP-dependent import, since ATP is required for translocation across the chloroplast envelope into the stroma (18) and in some cases for transport of lumenal proteins across the thylakoid membrane (19) . Following import termination, chloroplasts were divided into two fractions, and one fraction was treated with thermolysin.

The results from the import assays are shown in Fig. 3A. In the presence of light and ATP, import is evident, as judged by the disappearance of precursor and the appearance of a processed final product in both the nonprotease- and protease-treated light import assays (Fig. 3A, lanes4 and 5). The molecular weight of the processed product was estimated to be approximately 3 kDa, which agrees with the size of the mature product predicted previously from primary structure analysis of the PSII-T precursor. The absence of PSII-T precursor in the nonprotease-treated light import lane indicates that import occurred efficiently.


Figure 3: A, invitro protein import of PSII-T precursor into isolated pea chloroplasts. Lane1, initial translation product (TP); lane2, dark control minus thermolysin (D-); lane3, dark control plus thermolysin (D+); lane4, import in the light minus thermolysin (L-); lane5, import in the light plus thermolysin (L+); lane6, chloroplast soluble fraction (S); lane7, thylakoid membrane fraction minus thermolysin (M-); lane8, thylakoid membrane fraction plus thermolysin (M+); lane9, thylakoid membrane fraction plus thermolysin in Triton X-100 (M + TX). On the leftside: p, precursor; m, mature protein. 30 µl of D-, D+, L- and 15 µl of the S, L+, M-, M+, M + TX final pellet suspensions were precipitated with cold (-20 °C) acetone (95% final concentration) and centrifuged at 10,000 g for 10 min, and the pellets were dried and resuspended in 30 µl and 10 µl of SDS sample buffer, respectively. 10-µl aliquots were loaded in each lane. For D-, D+, L-, and S, lanes contain 10% of the chloroplasts or subfractions recovered from each assay. For L+, M-, M+, and M + TX, lanes contain 20% of the chloroplasts or subfractions recovered from each assay. B, treatment of PSII-T precursor with thermolysin in the absence of chloroplasts. Lane1, translation product; lane2, translation product plus thermolysin; lane3, translation product plus EDTA-treated thermolysin and SDS sample buffer.



Under dark conditions, import of substantial labeled PSII-T precursor is not observed, as judged by the presence of full-length PSII-T precursor in the nonprotease-treated import assays (Fig. 3A, lane2). The faint lower band suggests minimal import due to preexisting ATP in the reaction assay (ATP carried over from translation reactions, which was 50 µM or less, or endogenous chloroplast ATP). Upon treatment with protease, the precursor and several lower molecular weight bands are observed (Fig. 3A, lane3). The presence of protease-protected full-length precursor suggests that a portion of precursor molecules has completely entered the chloroplast without entering the stroma sufficiently for processing to occur. The presence of smaller size bands suggests that another portion of precursor molecules has been imported only partially and to various extents, the part extending outside the chloroplast envelope being degraded by the protease, the remaining part inside the chloroplast being protected. Protease-protected precursor is not a result of insensitivity to thermolysin since PSII-T treated with the protease was completely degraded, as shown in Fig. 3B, lane2. To exclude the possibility that residual thermolysin was still active after EDTA treatment and repurification of chloroplasts, the effectiveness of EDTA inactivation is shown in Fig. 3B, lane3. As can be seen, the PSII-T precursor treated with thermolysin and SDS sample buffer is not degraded after exposure to EDTA.

To determine the localization of the 3-kDa product, chloroplast fractionation experiments were performed. Import assays were carried out in the light as before, and after lysis, the chloroplasts were fractionated and the soluble fraction (S) was separated from the thylakoids by differential centrifugation. The thylakoid membrane fractions underwent three different treatments after imports and subfractionations: 1) not treated with protease (M-), 2) treated with protease (M+), 3) treated with protease after thylakoid membranes were disrupted with Triton X-100 (M + TX).

Radiolabeled protein is not detected in the soluble fraction (Fig. 3A, lane6). In the membrane fraction that was not treated with protease, the 3-kDa product is clearly present (lane7) and is also present in the protease-treated membrane fraction (lane8), indicating that the 3-kDa product is localized in the inner side of the thylakoid membrane and is therefore protected from protease action. Protease treatment after the membranes had been disrupted with Triton X-100, resulted in the disappearance of the 3-kDa band (lane9), demonstrating that the 3-kDa polypeptide is now sensitive to protease digestion and confirming that it is localized to the thylakoid lumen.

It is concluded from the results obtained from the chloroplast protein import experiments that the PSII-T precursor can be imported into pea chloroplasts in vitro and processed to a final product of 3 kDa, as the amino acid sequence analysis predicts. Furthermore, chloroplast fractionation experiments demonstrate that the mature PSII-T product is localized on the lumenal side of the thylakoid membrane.

Time Course Import Assays

It has been shown that the thylakoid lumen proteins OE33, OE23, and OE17 are targeted to the thylakoid lumen by a two step process, which includes the formation of a stromal intermediate after cleavage of the stroma targeting domain by the stromal protease (8, 10) . To examine whether the processing of PSII-T resembles that of the other lumenal proteins, time course import assays with PSII-T precursor were performed. Import reactions were conducted as before and were stopped at various time points soon after initiation of the import process by the addition of HgCl, which completely halts protein import into chloroplasts and all intrachloroplast activities (20) . Two sets of time course import assays were conducted, and one set was treated with protease after treatment with HgCl. Chloroplasts were reisolated, washed, and analyzed as described under ``Materials and Methods.'' The results of the nonprotease-treated time courses are shown in Fig. 4 (uppergel). As the PSII-T precursor disappears with time, two intermediate species appear, accumulate, and disappear, followed by the appearance and accumulation of the mature form. Time course import assays treated with protease (Fig. 4, bottomgel) showed a similar band pattern, confirming that these species are inside the chloroplast.


Figure 4: In vitro time course of protein importation of PSII-T. Import reactions were conducted in the presence of light and 10 mM ATP as described under Materials and Methods`` and terminated at the designated time points with HgCl. Uppergel, minus protease treatment of chloroplasts; bottomgel, plus protease treatment of chloroplasts. Final pellet suspensions from each time point were acetone-precipitated as described under ''Materials and Methods,`` and the equivalent of 5 µg of chlorophyll was loaded in each lane. On the left side, p, i1, i2, and m refer to precursor, intermediate 1, intermediate 2, and mature protein, respectively.



Under the conditions used, the import assay is partially a pulse-chase experiment. Continuous binding of new precursor molecules is not observed during the entire 10-min period, and intermediate molecular weight species disappear with time. However, the mature radioactive protein accumulates during the time course of the reaction, as shown by its higher than expected level of radioactivity. (There are 6 Met residues in the precursor and only 1 in the mature protein, yet equal amounts of chloroplast protein were loaded in the gel lanes.)

The larger intermediate species observed on the gels (i1), has a molecular mass of 7.5 kDa, similar to the molecular mass of the predicted intermediate containing the mature protein and the thylakoid targeting domain. The smaller intermediate (i2) was estimated to be approximately 3.8 kDa, close to the size estimated for both the predicted thylakoid targeting domain (4.3 kDa) and stroma targeting domain (3.57 kDa).

DISCUSSION

The results presented in this paper demonstrate that the nuclear cotton gene, psbT, encodes an 11-kDa protein that is the precursor of the PSII-T polypeptide, one of the low molecular weight proteins from photosystem II. PSII-T was first isolated from spinach PSII preparations as a 5-kDa polypeptide, and the N-terminal sequence was determined for 29 amino acids. A very high degree of homology exists between the spinach sequence and the C-terminal cotton PSII-T sequence. In cotton, PSII-T mature protein has a molecular weight of 3.0 kDa, as predicted by amino acid sequence analysis, making it the smallest polypeptide from PSII reported so far. It is possible that the spinach analogue is smaller than determined and its assigned size of 5 kDa is an overestimation, due to technical limitations of the SDS-PAGE of very small proteins.

In vitro protein import experiments with radiolabeled PSII-T precursor showed that the cotton PSII-T precursor is imported into isolated pea chloroplasts and that it is processed to the predicted 3.0-kDa mature form. Although the spinach PSII-T was isolated from PSII preparations of thylakoid membranes (2) , it was not determined whether the protein was facing the stromal or the lumenal side of the membrane. Chloroplast fractionation experiments presented here demonstrate that PSII-T is localized on the lumenal side of the thylakoid membrane, thus defining a new nuclear encoded lumenal protein. It may be a component of the oxygen-evolving complex that is known to reside in the thylakoid lumen (4) .

Time course import assays showed that maturation of the PSII-T precursor to its final form occurs through a two step process. First, a stromal intermediate is formed, which, upon translocation into the thylakoid membrane, is processed to the mature protein, consistent with the processing of other lumenal proteins (OE33, OE23, and OE17). The sizes of the stromal intermediate (Fig. 4, i1) and mature forms are those predicted from primary structure analysis of the PSII-T precursor. The second intermediate of approximately 3.8 kDa is transiently present during the time course importation (Fig. 4, i2). Since the exact stroma processing site is not known, it is difficult to assign this species to either the predicted stroma targeting or thylakoid targeting domain of 3.57 and 4.43 kDa, respectively. The fact that this species appears on the time course gels after the stromal intermediate suggests that it represents the thylakoid targeting domain. However, the identity of this species has not been rigorously established.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Current address: Dept. of Plant Sciences, University of Cambridge, Downing St., Cambridge CB2 3EA, United Kingdom.

Current address: Genetics Laboratory, UPR.CNRS 9026, Avenue des Facultes, 33405 Talence-Cedex, France.

**
To whom correspondence should be addressed. Tel.: 706-542-2086; Fax: 706-542-2090.

The abbreviations used are: PSII, photosystem II; PAGE, polyacrylamide gel electrophoresis; IB, import buffer.


ACKNOWLEDGEMENTS

We are very appreciative of the advice and assistance of the laboratory of Dr. K. Cline, University of Florida, and in particular Dr. R. Henry in carrying out the protein import experiments.


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