©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Polyprotein Precursor to the Euglena Light-harvesting Chlorophyll a/b-binding Protein Is Transported to the Golgi Apparatus Prior to Chloroplast Import and Polyprotein Processing (*)

Chidananda Sulli , Steven D. Schwartzbach (§)

From the (1) School of Biological Sciences, University of Nebraska, Lincoln, Nebraska 68588

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

The major Euglena thylakoid protein, the light harvesting chlorophyll a/b-binding protein of photosystem II (pLHCPII) is synthesized in the cytoplasm as a polyprotein precursor composed of a 141 amino acid presequence containing a signal peptide domain followed by eight mature LHCPIIs covalently linked by a decapeptide. To determine the transport route from cytoplasm to chloroplast and the site of polyprotein processing, Euglena was pulse labeled with [S]sulfate, organelles separated on sucrose gradients, and pLHCPII and LHCPII immunoprecipitated and separated on SDS gels. After a 10-min pulse, the pLHCPII polyprotein was found in the endoplasmic reticulum (ER) and Golgi apparatus. LHCPII was undetectable after a 10-min pulse consistent with the 20-min half-life for pLHCPII processing. When pulse-labeled cells were chased for 20 or 40 min with unlabeled sulfate, the fraction of pLHCPII in the ER decreased, and the fraction in the Golgi apparatus increased. LHCPII appeared only in thylakoids and chloroplasts, never in the ER or Golgi apparatus. NaCO extraction, a treatment that releases soluble but not integral membrane proteins, did not remove pLHCPII from ER and Golgi membranes. Trypsin digestion of ER and Golgi membranes produced 4 pLHCPII membrane protected fragments. The Euglena pLHCPII polyprotein is transported as an integral membrane protein from the ER to the Golgi apparatus and from the Golgi apparatus to the chloroplast. Polyprotein processing appears to occur during or soon after chloroplast import of the membrane-bound precursor.


INTRODUCTION

Transport of proteins into the ER() is co-translational and dependent upon an N-terminal extension, the signal sequence (1) . The nascent protein's signal sequence interacts with signal recognition particle, translation is arrested, the signal recognition particle-ribosome complex binds an integral ER membrane protein, elongation resumes, the nascent chain is translocated into the ER, and the signal sequence is cleaved (2, 3) . Integral membrane proteins are co-translationally inserted into the ER membrane in a linear manner (1) . A cleaved or uncleaved signal peptide initiates translocation, and a stop transfer sequence stops translocation anchoring the protein in the membrane (1, 4) . The orientation of proteins with multiple membrane-spanning domains is determined by the linear alternating appearance of start transfer membrane anchor sequences and stop transfer membrane anchor sequences (1, 5, 6). From the ER, proteins are transported to the Golgi apparatus where they are sorted for transport to their final destination (7) .

In contrast to protein transport into the ER, transport of proteins into chloroplasts is post-translational. Chloroplast proteins are synthesized in the cytoplasm as soluble precursors containing a N-terminal extension, the transit peptide, that interacts post-translationally with envelope receptors (8) . The transit peptide functions as a stroma targeting signal (9) and additional targeting information is required for localization within the chloroplast. LHCPII, the major thylakoid protein, spans the membrane three times, has a stromal targeting transit sequence that is not required for thylakoid integration (9, 10) , and the mature protein in a soluble form is integrated into the thylakoid (11) . Integration requires all three membrane-spanning domains (12, 13, 14) .

The pLHCPIIs of Euglena, a unicellular protist, are four proteins of 207, 161, 122, and 110 kDa encoded by 6.6- and 9.5-kilobase mRNAs (15, 16) . The sequence of two genomic clones indicates that the pLHCPIIs are polyproteins composed of multiple copies of LHCPII covalently joined by a decapeptide linker (16, 17) . Individual Euglena LHCPIIs are approximately 65% homologous to higher plant LHCPIIs containing the three hydrophobic membrane-spanning thylakoid insertion domains (16) . Euglena pLHCPII is synthesized on membrane-bound polysomes (18) as found for proteins translocated into the ER rather than on free ribosomes as found for higher plant and green algal pLHCPIIs. Euglena pLHCPII contains a 141-amino-acid N-terminal extension the first 35 amino acids of which have the characteristic features of a signal peptide (19) . Euglena pLHCPII is co-translationally inserted in vitro into canine microsomes and the signal peptide removed (19) . During times of active LHCPII synthesis, immunogold electron microscopy localizes Euglena LHCPII to the Golgi apparatus while at times when LHCPII is not being synthesized, an immunoreaction is only seen in the chloroplast (20, 21, 22) . In contrast to higher plant and green algal pLHCPIIs which are imported as soluble precursors directly into chloroplasts, Euglena pLHCPII appears to be co-translationally transported into the ER and transferred to the Golgi apparatus prior to chloroplast localization.

The ER and Golgi apparatus are the site of endoproteolytic processing of many proproteins and polyprotein precursors (23, 24, 25, 26) suggesting that pLHCPII polyprotein processing occurs in the ER or Golgi apparatus prior to chloroplast localization. Pulse-chase intracellular localization experiments were performed in order to delineate the intracellular route of pLHCPII transport to the chloroplast and identify the site of pLHCPII polyprotein processing. These experiments provide the first direct in vivo demonstration of the transport of Euglena pLHCPII from the ER to the Golgi apparatus prior to chloroplast localization. pLHCPII is transported as a membrane-bound polyprotein precursor whose endoproteolytic conversion into individual LHCPIIs occurs during or rapidly after chloroplast import. A brief report of this work has appeared (27) .


EXPERIMENTAL PROCEDURES

Cell Growth and Metabolic Labeling

Euglena gracilis Klebs var. bacillaris Cori maintained in our laboratory in the dark for many years was used throughout this work. Conditions for cell growth on low sulfur medium and preparation of dark grown resting cells were essentially as described (28, 29) . After 3 days on resting medium, cell division had ceased and chloroplast development was initiated by light exposure (28) . Growth on low sulfur medium has no effect on light induced chloroplast development (29) .

Cells (10 ml) exposed to light for 24 h were pulse-labeled with 600 µCi/ml of carrier free HSO (ICN Radiochemicals, Irvine, CA) by incubation at 26 °C with shaking in the light. A chase was initiated by the addition of KSO for a final concentration of 0.1 M. The addition of KSO immediately inhibited any further incorporation of [S]sulfate into trichloroacetic acid precipitable material (15) .

Cell Fractionation by Isopycnic Sucrose Gradient Centrifugation

All operations were performed at 0-4 °C. For measurements of marker enzyme activities, 250-ml cells were harvested by centrifugation at 1000 g for 5 min, washed with buffer A (25 mM HEPES-KOH, pH 7.4, 1 mM EDTA, and 400 mM sucrose) and resuspended in 1 ml of buffer A/g of cells. Cells were broken by grinding for 2.5 min with 2.5 g/ml acid-washed glass beads (250-300 µm, Sigma). Homogenates were removed from the settled beads and the beads washed three times with 0.5 ml of buffer A. The pooled homogenates were centrifuged at 150 g for 5 min to remove beads and unbroken cells. [S]Sulfate-labeled cells (10 ml) were harvested at room temperature by centrifugation at 3000 g for 2 min and resuspended in 0.5 ml of buffer A containing the protease inhibitors (Boehringer Mannheim) antipain (1 µg/ml), chymostatin (100 µg/ml), pepstatin (10 µg/ml), E64 (1 µg/ml), phenylmethylsulfonyl fluoride (0.5 mM), aprotinin (1 µg/ml), and leupeptin (1 µg/ml). Cells were broken by grinding with a glass pestile in a 15-ml plastic conical centrifuge tube (Nalgene) for 2.5 min with 1.0 g of glass beads. Homogenates were removed from the settled beads and the beads washed three times with 0.5 ml of buffer A. The pooled homogenates were centrifuged at 150 g for 5 min to remove beads and unbroken cells.

The clarified cell-free homogenate (1.5 ml) was loaded onto 12-ml gradients consisting of a 2-ml 20% (w/w) sucrose step on top of a 25-50% (w/w) linear sucrose gradient formed over a 0.5-ml cushion of 55% (w/w) sucrose. Gradients were centrifuged at 100,000 g (26,000 rpm) for 3 h in a Beckman SW 41 rotor and fractionated from the top into 0.4-ml fractions. Individual fractions were assayed for marker enzymes or precipitated with 10% trichloroacetic acid, and the trichloroacetic acid precipitates were solubilized prior to immunoprecipitation by boiling for 2 min in 100 µl of SDS buffer (2% SDS, 60 mM Tris-HCl, pH 8.6).

Assay for Membrane Integration by NaCO Extraction and Trypsin Digestion

A clarified cell-free homogenate (1.5 ml) prepared from 10 ml of pulse-labeled cells was centrifuged at 3,000 g for 10 min to obtain a 3,000 g chlorophyll-free supernatant and a 3,000 g pellet containing all of the chloroplast membranes. The pellet was resuspended in buffer A while the supernatant was used directly for NaCO extraction and protease protection assays. NaCO and potassium acetate extraction were performed by adjusting a 0.5-ml aliquot of the 3000 g pellet and supernatant fractions to 0.1 M NaCO pH 11.5 or 0.5 M potassium acetate, incubating on ice for 30 min, and recovering the extracted membranes by centrifugation for 1 h at 150,000 g in a Beckman Ti-70.1 rotor. The 150,000 g membrane pellet resuspended in buffer A, and the supernatants were precipitated with 10% trichloroacetic acid (w/v). The trichloroacetic acid precipitates were solubilized prior to immunoprecipitation by boiling for 2 min in 100 µl of SDS buffer.

Protease protection assays were performed by incubating the 3,000 g supernatant on ice for 30 min with 0.1 mg/ml trypsin (Type XIII, Sigma) in the presence or absence of 0.5% Triton X-100. An aliquot was incubated in the absence of trypsin as a control for endogenous proteolysis. Trypsin was added from a 1 mg/ml stock solution prepared in 50 mM HEPES-KOH, pH 7.5, 100 mM NaCl, 0.2 M sucrose. Proteolysis was terminated by addition of phenylmethylsulfonyl fluoride for a final concentration of 10 mM and the membranes recovered by centrifugation for 1 h at 150,000 g in a Beckman Ti-70.1 rotor. The membrane pellet was solubilized prior to immunoprecipitation by boiling for 2 min in 100 µl of SDS buffer.

Immunoprecipitation and SDS-Gel Electrophoresis

Immunoprecipitation with anti-LHCPII and protein A-Sepharose (Pharmacia LKB Biotechnology) was performed as described previously (15) . Immunoprecipitated proteins were separated on 8-12% linear SDS-polyacrylamide gels (30) or 12% Tricine gels (31) . Gels were impregnated with 1 M sodium salicylate (32), dried, and exposed to preflashed Kodak X-Omat AR film at -70 °C. Radioactivity in individual bands was quantified with a PhosphorImager (Molecular Dynamics).

Analytical Methods

Rotenone-insensitive NADH-cytochrome c reductase activity was measured at 25 °C by following the increase in absorbance at 550 nm (33) . The 1-ml reaction mixture contained a 100-µl gradient fraction, 50 mM potassium phosphate, pH 7.5, 40 µM cytochrome c, 1 mM potassium cyanide, and 1.5 µM rotenone. Glucose-6-phosphatase activity was assayed at 30 °C by the method of Arion (34) . The 100-µl reaction mixture contained a 30-µl gradient fraction, 50 mM sodium cacodylate buffer, pH 6.5, and 20 mM glucose 6-phosphate (disodium salt, Sigma). After incubation for 30 min, the reaction was stopped by addition of 0.9 ml of stop solution composed of 0.42% ammonium molybdate in 1 N HSO, 10% SDS, 10% ascorbic acid (6:2:1, v/v/v), and the amount of inorganic phosphate released was measured by absorption at 720 nm. Latent IDPase was determined at 30 °C by the method of Bowles and Kauss (35) . The 100-µl reaction mixture contained a 15-µl gradient fraction, 60 mM Tris, pH 7.4, 5 mM IDP, and 1 mM MgCl with and without 0.1% (v/v) Triton X-100. After incubation for 20 min, the reaction was stopped by addition of 0.9 ml of stop solution, and the amount of inorganic phosphate released was measured by absorption at 720 nm. Chlorophyll was determined by measuring autofluorescence (36) .


RESULTS

Separation of Euglena Subcellular Organelles by Isopycnic Sucrose Gradient Centrifugation

In vivo studies of the intracellular transport and post-translational proteolytic processing of pLHCPII required the development of a rapid method for isolating organelles from small samples of pulse labeled cells. A number of methods have been developed for preparing Euglena extracts containing functional intact organelles. The method of choice is to digest the pellicle for 1 h with trypsin followed by osmotic disruption (37). The long time (1 h) required for pellicle weakening precludes using trypsinization to obtain subcellular organelles for pulse-chase localization studies. An alternative to osmotic lysis of trypsinized Euglena is to break cells by grinding with glass beads (38) . By modifying a previously published grinding procedure (38) , a rapid method for preparing crude cell-free extracts containing 40-60% intact chloroplasts was developed. Within 5-7 min of harvest, crude cell-free extracts could be prepared from pulse-labeled cells and loaded onto sucrose gradients for isopycnic separation of subcellular organelles.

Organelle-specific marker enzyme distributions were used to determine the location of specific subcellular organelles on isopycnic sucrose density gradients. Chlorophyll, a marker for thylakoid membranes and intact chloroplasts, formed two peaks between fractions 15-18 and 19-23 (Fig. 1). The small subunit of ribulose bisphosphate carboxylase (SSU), a soluble chloroplast protein (38) , formed a peak coincident with the chlorophyll peak in fractions 19-23 (Fig. 1) localizing intact chloroplasts to this region. SSU was not found in the low density chlorophyll peak, fractions 15-18 (Fig. 1), indicating that these fractions contain thylakoid membranes. The SSU peak in fractions 1-5 at the top (Fig. 1) of the gradient is protein released from broken chloroplasts.


Figure 1: Localization of Euglena organelles on isopycnic sucrose density gradients. A crude cell homogenate (1.5 ml) prepared from dark-grown Euglena exposed to light for 24 h was loaded onto a 12-ml gradient consisting of a 2-ml 20% (w/w) sucrose step on top of a 25-50% (w/w) linear sucrose gradient formed over a 0.5-ml cushion of 55% (w/w) sucrose. Gradients were centrifuged at 100,000 g (26,000 rpm) for 3 h in a Beckman SW 41 rotor and fractionated from the top into 0.4-ml fractions. Organelles were identified by measuring the following activities: chlorophyll, a marker for thylakoids and intact chloroplasts; SSU, a marker for intact chloroplasts and soluble fraction (released from broken chloroplasts); rotenone-insensitive NADH cytochrome c reductase (NADH cyt c red), an ER and mitochondrial marker; glucose-6-phosphatase (Glc-6-Pase), an ER marker; latent IDPase, a Golgi marker.



Latent IDPase, a Golgi marker enzyme (39) , formed two peaks between fractions 10-18 (Fig. 1). The second IDPase peak was always less dense and resolved from thylakoid membranes. There was no overlap between Golgi membrane peaks and intact chloroplasts. Golgi membranes from Euglena(39) and other algae (40, 41) are often resolved into multiple fractions of different density. It is not known whether these different density fractions represent functionally distinct Golgi compartments.

Glucose-6-phosphatase, localized exclusively in the Euglena ER (38) , formed a single peak in fractions 3-6 (Fig. 1). Rotenone-insensitive NADH cytochrome c reductase, localized in both the Euglena ER and outer mitochondrial membrane (42), formed a peak in fractions 3-6 (Fig. 1) confirming that ER membranes are localized to this region of the gradient. The glucose-6-phosphatase, NADH cytochrome c reductase ER peak was resolved, albeit poorly, from the peak of solubilized SSU remaining at the top of the gradient (Fig. 1).

The major peak of rotenone-insensitive NADH cytochrome c reductase was in fractions 20-23 (Fig. 1). Succinate dehydrogenase,() a mitochondrial inner membrane protein, was found in fractions 20-23 identifying them as containing intact mitochondria. A minor peak of rotenone-insensitive NADH cytochrome c reductase was found in fractions 8-11 between the ER and Golgi membranes (Fig. 1). Glucose-6-phosphatase was not present in these fractions eliminating the possibility that they contain ER membranes. Succinate dehydrogenase was not found in fractions 8-11. The presence of NADH cytochrome c reductase and absence of succinate dehydrogenase from fractions 8-11 suggests that mitochondrial outer membranes band in this region of the gradient.

Intracellular Transport and Subcellular Site of pLHCPII Polyprotein Processing

Euglena pLHCPIIs are a group of extremely large polyproteins having molecular masses of 110, 122, 161, and 207 kDa (15) that are converted to LHCPIIs with a 20-min half-life (15) . When cells exposed to light for 24 h were pulse labeled for 10 min with [S]sulfate, the pLHCPIIs are the only proteins immunoprecipitated (Fig. 2A). The 26- and 27-kDa mature LHCPIIs (15) are undetectable. After a 20-min chase with unlabeled sulfate, pLHCPIIs and mature LHCPIIs are found in the immunoprecipitate (Fig. 2B) while after a 40-min chase, mature LHCPIIs are the predominant proteins immunoprecipitated (Fig. 2C). In order to determine the intracellular route of pLHCPII transport from the cytoplasm to the chloroplast and the intracellular site of polyprotein processing, cell-free extracts were prepared from [S]sulfate-labeled cells, organelles were separated by isopycnic density gradient centrifugation, and pLHCPII and LHCPII were immunoprecipitated from each fraction and separated by SDS gel electrophoresis.


Figure 2: Intracellular localization of pLHCPII and LHCPII. Dark-grown Euglena exposed to light for 24 h were pulse labeled for 10 min with [S]sulfate. A chase was initiated by addition of unlabeled sulfate for a final concentration of 0.1 M. Cell-free extracts were prepared at the end of the 10-min pulse (A), 20 (B), or 40 min © after the start of the chase. Organelles were separated by isopycnic centrifugation on 12-ml gradients consisting of a 2 ml 20% (w/w) sucrose step on top of a 25-50% (w/w) linear sucrose gradient formed over a 0.5-ml cushion of 55% (w/w) sucrose. After centrifugation at 100,000 g (26,000 rpm) for 3 h in a Beckman SW 41 rotor, gradients were fractionated from the top into 0.4-ml fractions. Each fraction was precipitated with 10% trichloroacetic acid, resuspended by boiling in 2% SDS, and an aliquot was immunoprecipitated with antibody to Euglena LHCPII. An aliquot of the crude cell-free extract loaded onto the gradient was also immunoprecipitated. The immunoprecipitates were analyzed on 8-12% linear gradient Tris-glycine SDS-polyacrylamide gels and the immunoprecipitated proteins visualized by fluorography. The regions of the fluorographs containing pLHCPII and LHCPII are presented.



The subcellular localization of LHCPII was distinctly different from pLHCPII. (Fig. 2). pLHCPIIs were found predominately in the ER, fractions 3-6, and Golgi apparatus, fractions 10-18, while LHCPII was found predominately in thylakoids, fractions 15-18, and chloroplasts, fractions 19-22 (Fig. 2). All four pLHCPIIs were found in each pLHCPII containing fraction. ER localized pLHCPIIs were degraded while pLHCPIIs in other regions of the gradient were relatively undegraded (Fig. 2). Organelle breakage occurred during cell disruption releasing organelle proteases into the soluble cytoplasmic fraction. Degradation of ER localized pLHCPII probably results from incomplete separation of the ER fraction from the protease containing soluble fraction remaining at the top of the gradient. The inclusion of protease inhibitors in the isolation and gradient buffers did not fully inhibit pLHCPII degradation.

Large differences exist in the amount of pLHCPII and LHCPII found in each gradient fraction (Fig. 2). These differences exceed the linear range of fluorographic film response. The fluorographic image therefore does not provide a quantitative visual representation of the amount of pLHCPII and LHCPII in subcellular organelles. To overcome this problem, storage phosphorimaging which has a large linear response range was used to accurately measure the amounts of pLHCPII and LHCPII in each fraction. The SDS gels used for the fluorographs presented in Fig. 2were scanned with a PhosphorImager. pLHCPII levels are defined as the sum of the image intensity for the 207-, 161-, 122-, and 110-kDa pLHCPIIs since the distribution of each of the four major pLHCPIIs was identical to the total pLHCPII distribution. LHCPII levels are defined as the sum of the image intensity for the 26- and 27-kDa LHCPIIs. The total S-labeled protein did not change during the chase, but the extent of cell breakage and thus the fraction of total cellular S-labeled protein in the homogenate varied from sample to sample. To allow direct comparisons between gradients loaded with differing amounts of S-labeled protein, the amount of pLHCPII and LHCPII in each fraction is plotted in Fig. 3as the percent of total immunopreciptate (pLHCPII and LHCPII) recovered from the gradient.


Figure 3: Quantitative analysis of pLHCPII and LHCPII intracellular localization. The SDS gels presented in Fig. 2 from a 10-min pulse (A), a 20-min (B) or 40-min chase were scanned with a PhosphorImager. pLHCPII levels are defined as the sum of the image intensity for the 207-, 161-, 122-, and 106-kDa pLHCPIIs and LHCPII levels are defined as the sum of the image intensity for the 26- and 27-kDa LHCPIIs. To allow direct comparisons between gradients loaded with differing amounts of S-labeled protein, the amount of pLHCPII and LHCPII in each fraction is plotted as a percent of the total immunoprecipitate (pLHCPII and LHCPII) recovered from the gradient.



After a 10-min pulse, a pLHCPII peak was found in fractions 3-6, ER membranes, a shoulder in fractions 8-12, low density Golgi membranes, and a second peak in fractions 13-18, high density Golgi membranes (Fig. 2A and 3A). The distribution of pLHCPII between the low and high density Golgi fractions varied from experiment to experiment while the total fraction of Golgi localized pLHCPII remained relatively constant. The major pLHCPII peaks were less dense and resolved from the two chlorophyll peaks used as internal standards to locate thylakoids and chloroplasts (Fig. 3A). Extensive degradation of ER localized pLHCPII (Fig. 2) results in a large fraction of immunoprecipitable radioactivity having molecular weights differing from that of the four pLHCPIIs. These degraded pLHCPIIs are not resolved as distinct polypeptides and could not be quantitated by the software employed resulting in a significant underestimate of ER localized pLHCPII. The reported amount (Fig. 3) of ER localized pLHCPII is thus a minimum estimate. The absence of degradation (smearing) in other gradient regions allows accurate determinations of Golgi, thylakoid, and chloroplast localized pLHCPII (Fig. 3).

pLHCPII was present but did not form a distinct peak in thylakoids and chloroplasts ( Fig. 2and Fig. 3). This material could either represent thylakoid and chloroplast localized pLHCPII or ER and Golgi localized pLHCPII that associates with chloroplast membranes during subcellular fractionation. Chlorophyll provides a direct measure of the amount of chloroplast membranes in each gradient fraction from the 10-min pulse (Fig. 3A). If pLHCPII was transported into chloroplasts and accumulated in thylakoids prior to processing, the amount of pLHCPII in chloroplasts and thylakoids would be proportional to the amount of chlorophyll recovered in the respective fractions. Chloroplasts from the 10-min pulse-labeled sample, fractions 19-22, contained more chlorophyll than thylakoids, fractions 15-18 (Fig. 3A). Chloroplasts, however, contained significantly less pLHCPII than thylakoids (Fig. 3A). The pLHCPII in the thylakoid region of the gradient is probably Golgi localized material that has been trapped by chloroplast membranes during centrifugation and not pLHCPII accumulating in chloroplasts and thylakoids prior to processing.

The fraction of total immunoprecipitate recovered as pLHCPII in the ER, fractions 3-6, decreased and the fraction in the Golgi apparatus, fractions 10-18, increased during a 20 or 40 min chase (Fig. 2, B and C, and 3 B and C). Partially processed pLHCPIIs were not detected on the gradient (Fig. 2, B and C). At the end of a 10-min pulse, 20% of pLHCPII was in the ER and 70% in the Golgi apparatus. After a 20-min chase, only 12% of pLHCPII was in the ER while 85% was in the Golgi apparatus. By the end of the 40-min chase, 12% of pLHCPII was ER localized and 70% was Golgi localized. Although the distribution of pLHCPII between the ER and Golgi varied from experiment to experiment, the fraction of ER localized pLHCPII was always significantly higher at the end of a 10-min pulse than after a 20- or 40-min chase.

The subcellular localization of LHCPII was distinctly different from pLHCPII ( Fig. 2and Fig. 3). LHCPII was not present in the ER, fractions 3-6. The disappearance of pLHCPII during a 20- or 40-min chase was associated with the appearance of LHCPII in thylakoids, fractions 15-18, and chloroplasts, fractions 19-22 (Fig. 3, B and C). The LHCPII thylakoid peak was clearly distinct from but overlapped the pLHCPII Golgi peak (Fig. 2, B and C and 3, B and C). The absence of LHCPII from the ER and most pLHCPII containing Golgi fractions suggests that pLHCPII is transferred as a polyprotein from the ER and Golgi apparatus to the chloroplast where during or rapidly after chloroplast import, the pLHCPII polyprotein is cleaved into mature LHCPIIs. Due to overlap between Golgi and thylakoid membrane containing fractions, the possibility can not be eliminated that processing occurs in a Golgi subcompartment having the same density as thylakoids but totally distinct from the other Golgi containing fractions.

The Euglena pLHCPII Polyprotein Is Transported to the Chloroplast as an Integral Membrane Protein

Euglena pLHCPII synthesized in vitro is co-translationally inserted into canine microsomes, processed by signal peptidase, and anchored within the membrane remaining protease accessible on the cytoplasmic membrane face (19) . Alkaline NaCO extraction converts membrane vesicles into open membrane sheets releasing luminal and peripheral proteins (43) . NaCO extraction and protease protection assays have been used to determine whether in vivo synthesized pLHCPII is an integral ER and Golgi protein with protease accessible cytoplasmic domains as suggested by the in vitro processing studies.

Crude cell-free extracts were fractionated into a 3000 g pellet containing 87% of the chlorophyll, 14% of the IDPase, and 40% of the glucose-6-phosphatase activity and a 3000 g supernatant containing only 3% of the chlorophyll, 90% of the IDPase, and 60% of the glucose-6-phosphatase activity. Based on the marker enzyme distribution, the 3000 g pellet contains thylakoids, chloroplasts, and ER membranes while the 3000 g supernatant is a chloroplast membrane-free ER and Golgi fraction. Since gradient fractionation localized pLHCPII to the ER and Golgi apparatus with little in thylakoids and chloroplasts (Fig. 3), the 3000 g pellet was used to study the topology of ER localized pLHCPII. Membranes often aggregate during pelleting sequestering proteins within the aggregate making them inaccessible to exogenous protease (44) . Possible aggregation artifacts were avoided by using the 3000 g supernatant as an ER-Golgi membrane fraction.

Extraction of the 3000 g pellet and supernatant with 0.5 M potassium acetate or 0.1 M NaCO did not remove pLHCPII from ER and Golgi membranes (Fig. 4A). All four pLHCPIIs were recovered in the acetate and NaCO extracted membrane pellet, and pLHCPIIs were barely detectable in the membrane-free supernatant (Fig. 4A). PhosphorImager analysis of the gels indicated that 95% of the two larger pLHCPIIs and 87% of the two smaller pLHCPIIs remained membrane associated after the 3000 g pellet was extracted with potassium acetate. Extraction of the 3000 g supernatant with potassium acetate resulted in 75% of the two larger and 89% of the two smaller pLHCPIIs remaining membrane-associated. NaCO extraction of the 3000 g pellet resulted in 95% of the two larger and only 64% of the two smaller pLHCPIIs remaining membrane associated while 78% of the two larger and only 56% of the two smaller pLHCPIIs were recovered in the membrane pellet after extracting the 3000 g supernatant with NaCO. The recovery of pLHCPII with NaCO extracted ER and Golgi membranes identifies pLHCPII as an integral membrane protein.


Figure 4: Potassium acetate and NaCO extractability of organelle-associated pLHCPII, LHCPII, and SSU. Euglena exposed to light for 24 h was pulse labeled for 10 min with [S]sulfate (A) or for 30 min with [S]sulfate (B). A cell-free extract was centrifuged at 3000 g for 10 min producing a chloroplast and ER membrane containing 3000 g pellet and a chlorophyll-free ER and Golgi membrane containing 3000 g supernatant. A, the resuspended 3000 g pellet (lanes 1-5) and supernatant (lanes 6-10) were adjusted to 0.5 M potassium acetate (Ac) or 0.1 M NaCO, pH 11.5 (CO), the extracted membranes recovered by centrifugation, and pLHCPII in the membrane pellet (P) and supernatant (S) was immunoprecipitated. B, the resuspended 3000 g pellet (lane 1) was adjusted to 0.5 M potassium acetate (Ac, lanes 2 and 3) or 0.1 M NaCO, pH 11.5 (COlanes 4 and 5), the extracted membranes recovered by centrifugation and LHCPII and SSU in the membrane pellet (P) and supernatant (S) were immunoprecipitated. Immunoprecipitates were analyzed on 8-12% (pLHCPII and LHCPII) or 8-16% (SSU) linear gradient Tris-glycine SDS-polyacrylamide gels. The regions of the fluorographs containing pLHCPII (A), LHCPII (B), and SSU (B) are presented.



To confirm that NaCO extraction can distinguish soluble from integral membrane proteins, the extractability of the small subunit of ribulose bisphosphate carboxylase (SSU), a soluble chloroplast protein, and LHCPII, an integral thylakoid membrane protein was determined (Fig. 4B). A lack of characterized ER and Golgi localized Euglena proteins necessitated using chloroplast proteins for these experiments. SSU was recovered in the membrane pellet when chloroplasts in the 3000 g pellet were extracted with potassium acetate and SSU was recovered in the membrane-free supernatant after extracting chloroplasts with 0.1 M NaCO (Fig. 4B). LHCPII on the other hand was recovered in the membrane pellet when chloroplasts were extracted with either potassium acetate or NaCO (Fig. 4B). The removal of SSU but not LHCPII from chloroplasts by NaCO extraction demonstrates that under the conditions used in these experiments, NaCO extractability distinguishes soluble proteins from integral membrane proteins.

Recovery of in vivo synthesized pLHCPII in the membrane fraction after NaCO extraction demonstrated that pLHCPII is anchored in the ER and Golgi membrane. Protease protection assays were performed in order to obtain information about the topology of pLHCPII within the membrane. pLHCPIIs were not degraded by endogenous proteases during incubation of the 3000 g supernatant on ice (Fig. 5, A and B). Trypsin digested all four pLHCPIIs. Four polypeptides, 24, 22, 15, and 10 kDa were recovered with trypsin-digested membranes only in the absence of detergent demonstrating that they are membrane protected digestion products (Fig. 5, A and B). Immunoprecipitable peptides were undetectable in the membrane-free supernatant. Analysis of the digestion products on Tricine gels did not detect additional low molecular weight protease protected fragments (Fig. 5B). Trypsin digestion of the 3000 g pellet produced the same protected fragments as obtained from digestion of the 3000 g supernatant. Two of the protected fragments, 24 and 22 kDa, are similar in size to thylakoid-protected LHCPII trypsin digestion products. Taken together, the protease protection and NaCO extraction experiments indicate that multiple membrane-spanning regions anchor pLHCPII in the ER and Golgi membrane.


Figure 5: Trypsin digestion of ER and Golgi localized pLHCPII. Euglena exposed to light for 24 h was pulse labeled for 10 min with [S]sulfate. A chlorophyll-free ER and Golgi membrane containing 3000 g supernatant (lane 1) was digested with trypsin (0.1 mg/ml) in the absence or presence of 0.5% Triton X-100. Proteolysis was terminated by addition of phenylmethylsulfonyl fluoride for a final concentration of 10 mM and the membranes recovered by centrifugation. pLHCPII-protected fragments were immunoprecipitated and separated on 8-12% linear gradient Tris-glycine SDS-polyacrylamide gels (A) or 12% Tricine SDS gels (B) and the immunoprecipitated peptides visualized by fluorography.




DISCUSSION

Immunoelectron microscopy provided the first indication that Euglena has a unique mechanism for chloroplast protein import. Upon exposure of dark-grown resting Euglena to light, anti-LHCPII immunogold labeling is first detected over the COS (20, 22, 45), a cytoplasmic osmiophilic structure composed of a central osmiophilic core with connecting reticulate extensions creating a compartment containing cytoplasmic material including ribosomes (20, 22, 45, 46) . As chloroplast development proceeds, labeling is detected over the Golgi apparatus and finally the chloroplast. The chloroplast is the only organelle labeled in cells that are not actively synthesizing LHCPII (20, 21, 22) .

In vivo and in vitro studies complementing the immunomicroscopy have further elucidated the novel mechanism of chloroplast protein import used by Euglena. pLHCPII is synthesized on membrane-bound polysomes rather than free polysomes, and the presequence 35 amino acid signal peptide domain is removed during co-translational insertion into canine microsomal membranes (18, 19) . At the end of a 10-min pulse, subcellular fractionation localizes pLHCPII to the ER and Golgi apparatus. Little chloroplast localized pLHCPII is seen at the end of the pulse, and mature LHCPII is undetectable. During a 20- or 40-min chase, the fraction of ER localized pLHCPII decreases, Golgi localized pLHCPII increases, and mature LHCPII but not pLHCPII accumulates in the chloroplast and thylakoid fractions. Diverse experimental approaches thus provide evidence for Euglena pLHCPII co-translational signal peptide-dependent transport to the ER and from the ER to the Golgi apparatus prior to chloroplast localization. This contrasts with the direct post-translational chloroplast uptake of plant and green algal pLHCPII.

NaCO did not remove pLHCPII from Euglena ER and Golgi membranes demonstrating it is an integral membrane protein. Trypsin digestion produced four protease-protected pLHCPII fragments. The individual LHCPIIs within the polyprotein are highly homologous (16, 17) . Trypsin cleavage sites are found at similar positions within each mature LHCPII unit of the polyprotein. Each protease-protected fragment may be derived from more than one LHCPII unit. The two largest protease-protected fragments (22 and 24 kDa) are similar in size to the protected fragments produced by trypsin digestion of Euglena thylakoids. Higher plant LHCPIIs are inserted into thylakoids post-translationally and the three membrane-spanning domains orient the protein with the N terminus in the stroma and C terminus in the lumen (12, 13, 14, 47) . Euglena and higher plant LHCPIIs are not inserted into microsomal membranes or bacterial inner membranes (13, 19, 48) . A chimeric plant LHCPII containing a bacterial signal peptide at its N terminus is, however, inserted into the bacterial inner membrane and anchored within the membrane by hydrophobic sequences within LHCPII (48) . Signal peptide initiated linear insertion of the chimera into the membrane appears to enable one or all of the hydrophobic LHCPII domains to be inserted within the membrane. Euglena pLHCPII is co-translationally inserted into canine microsomal membranes, and signal peptidase removes the first 35 N-terminal amino acids of the presequence (19) demonstrating that it is a naturally occurring signal peptide-LHCPII chimera. Based on the similarity in size between the two largest pLHCPII ER-Golgi-protected fragments and the LHCPII thylakoid-protected fragments, it is tempting to speculate that some if not all LHCPII units within the pLHCPII polyprotein are anchored within the ER and Golgi membrane in the same orientation as in thylakoids, spanning the membrane three times with the N terminus in the cytoplasm and the C terminus in the lumen.

The ER and Golgi apparatus are sites of endoproteolytic processing of many proproteins and polyproteins (23, 24, 25, 26) . Some Semliki Forest Virus proteins are produced by signal peptidase cleavage of a polyprotein precursor (23) . Polyprotein precursors to neuroendocrine peptide hormones, yeast mating factor, and some proproteins are processed by trans-Golgi network resident proteases (24, 25, 26) . Vacuolar protein processing is, however, completed by sequence-specific vacuole resident proteases (49) .

pLHCPII polyprotein maturation requires endoproteolytic removal of the presequence and the conserved decapeptide linker joining individual LHCPIIs within the polyprotein. Pulse-chase experiments showed that LHCPII was localized only in chloroplasts and thylakoids, never in the ER and Golgi apparatus. pLHCPII was predominately ER and Golgi localized. Distinct processing intermediates, immunoprecipitable proteins with a molecular mass less than the smallest pLHCPII (116 kDa), were not seen in the Golgi apparatus during the chase. Unlike maturation of many proproteins and polyprotein precursors, the pLHCPII signal peptide-like decapeptide linker is not cleaved by signal peptidase in the ER or by Golgi resident proteases. As found for vacuole proteins, pLHCPII appears to be processed to LHCPII upon reaching its final intracellular location, the chloroplast (49) .

LHCPII is one of many Euglena chloroplast proteins synthesized in the cytoplasm and transported to the chloroplast. Amino acid sequences for five Euglena precursors have been deduced from cDNA sequences (19, 50, 51, 52, 53) . The precursor presequences differ but contain common structural features. Four are about 140 amino acids long, they are enriched in hyroxylated amino acids, they have a signal peptide domain with a potential signal peptidase cleavage site 35-50 amino acids from the N terminus, and they contain at least one additional hydrophobic domain that could function as a membrane anchor-stop transfer sequence (19, 50, 51, 52) . The oxygen evolving enhancer protein presequence is shorter, 93 amino acids, contains two hydrophobic domains, but lacks a N-terminal signal peptide (53) . Trans-splicing adds a 5` end to all Euglena mRNAs (54) . The oxygen evolving protein cDNA lacks this trans-spliced 5` end (53) indicating it is not full-length. The smaller size and absence of a N-terminal signal peptide is thus explained by its being deduced from an incomplete cDNA. The common structural framework of the Euglena presequences, especially the signal peptide domain, suggests that all cytoplasmically synthesized chloroplast proteins are transported as integral membrane proteins from the ER to the Golgi apparatus and from the Golgi apparatus to the chloroplast.

In contrast to green algae and higher plant chloroplasts which are surrounded by two membranes, Euglena chloroplasts are surrounded by three membranes suggesting that they evolved from a eukaryotic rather than a prokaryotic endosymbiont (55) . It has been postulated that the third chloroplast membrane evolved from the plasma membrane of the original photosynthetic symbiont, that it evolved from a phagocytic vacuole membrane, or that it is part of the ER (55) . Utilization of the secretory pathway for protein transport from the cytoplasm to the chloroplast is not surprising when one considers the evolutionary origin of the third membrane surrounding Euglena chloroplasts.

How integral membrane precursor proteins are transferred from Golgi transport vesicles through the three membranes surrounding Euglena chloroplasts remains unknown. Transformation of the Euglena proplastid into a chloroplast requires lipids for envelope expansion and thylakoid formation. The Euglena mutant WBUL lacks most if not all of the chloroplast genome but contains a proplastid remnant that upon light exposure undergoes limited development (56) suggesting a cytoplasmic origin for chloroplast lipid. Electron microscopy found membrane aggregates in the form of membrane whorls and osmiophilic bodies in the cytoplasm of dark grown Euglena(56, 57, 58) . Shortly after initiation of proplastid development, the membrane whorls are seen extending from the interior of the prolammellar body, the osmiophilic bodies are fused to the proplastid envelop, and the osmiophilic membrane aggregates are dispersed in the stroma (57, 58) . Since protease protection assays suggest that pLHCPII is integrated into the Golgi membrane in the same orientation as found within the thylakoid, it is tempting to speculate that Euglena thylakoid assembly is initiated within the ER and Golgi apparatus. Interaction between precursor proteins or their presequences within the Golgi transport vesicles could cause vesicle aggregation producing the membrane whorls observed by electron microcopy. Internalization of these precursor containing transport vesicles through fusion with the proplastid envelop membrane, release of stromal proteins from the internalized vesicles by presequence cleavage, migration of thylakoid membrane proteins within the lipid bilayer, and integration of chloroplast synthesized thylakoid proteins into the developing thylakoid would complete thylakoid assembly initiated within the ER and Golgi apparatus. Although highly speculative, this model is consistent with ultrastructural and biochemical studies of Euglena chloroplast biogenesis providing a mechanism for chloroplast import of membrane bound precursors.


FOOTNOTES

*
This work was supported by National Science Foundation Grant MCB-9118721 and funds from the University of Nebraska Center for Biotechnology. 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.

§
To whom correspondence and reprint requests should be addressed: 303 Lyman Hall, University of Nebraska, Lincoln, Nebraska 68588-0343. Tel.: 402-472-1682; Fax: 402-472-8722.

The abbreviations used are: ER, endoplasmic reticulum; COS, cytoplasmic osmiophilic structure; LHCPII, light harvesting chlorophyll a/b-binding protein of photosystem II; pLHCPII, precursor to the light harvesting chlorophyll a/b-binding protein of photosystem II; pSSU, precursor to the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase; SSU, small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase; Tricine, N-tris[hydroxymethyl]-methylglycine.

C. Sulli and S. D. Schwartzbach, unpublished observations.


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