Analysis of apicoplast targeting and transit peptide processing in Toxoplasma gondii by deletional and insertional mutagenesis

Molecular & Biochemical Parasitology 118 (2001) 11 – 21 www.parasitology-online.com.

Analysis of apicoplast targeting and transit peptide processing in Toxoplasma gondii by deletional and insertional mutagenesis Sunny Yung, Thomas R. Unnasch *, Naomi Lang-Unnasch Di6ision of Geographic Medicine, BBRB 203, Uni6ersity of Alabama at Birmingham, 1530 3rd A6enue South, Birmingham, AL 35294 -2170, USA Received 22 June 2001; accepted in revised form 14 August 2001

Abstract Deletion and insertion mutagenesis was used to analyze the targeting sequence of the nuclear encoded apicoplast protein, the ribosomal protein small subunit 9 of Toxoplasma gondii. Previous studies have shown that nuclear encoded apicoplast proteins possess bipartite leaders having characteristic signal sequences followed by serine/threonine rich transit sequences. Deletion analysis demonstrated that the first 55 amino acids of the rps9 leader were sufficient for apicoplast targeting. Insertional mutagenesis tagging the leader sequence with a hemagglutinin (HA) tag was used to study the events involved in the targeting pathway. Transfectants with insertions near the N-terminus of the transit displayed HA tagged precursors outside of the apicoplast, in the perinuclear region. In contrast, transfectants with the HA tag inserted near the carboxyl end of the transit-like region had apicoplast labeling. Western blot analysis of HA tagged stable isolates suggested that processing of the HA tagged leaders was a multi-step process, with processing occurring both outside of and at or within the apicoplast. © 2001 Elsevier Science B.V. All rights reserved. Keywords: Toxoplasma gondii; Apicoplast; Protein targeting; Ribosomal protein

1. Introduction The apicomplexan apicoplast is a non-photosynthetic plastid that is essential for cell survival [1]. First identified in Plasmodium and Toxoplasma [2 – 4], the apicoplast now appears widespread throughout the phylum [5]. Ultrastructural and molecular data suggest this organelle originated by secondary endosymbiosis, whereby a heterotrophic eukaryote engulfed and retained a photosynthetic eukaryote, resulting in a plastid with more than two membranes [6]. Electron microscopy studies have shown the apicoplast in Toxoplasma gondii to be surrounded by four membranes [4], two inner membranes from the plastid membranes of the primary endosymbiont, a third membrane hypotheAbbre6iations: GFP, green fluorescent protein; HA tag, hemagglutinin tag; rps9, ribosomal protein small subunit 9. * Corresponding author. Tel.: + 1-205-975-7601; fax: +1-205-9335671. E-mail address: [email protected] (T.R. Unnasch).

sized to be the plasma membrane of the secondary endosymbiont and an outermost membrane that appears to be connected to the host endomembrane system. Protein trafficking to secondary or complex plastids is complicated by these extra membranes. Thus, nuclear encoded proteins destined for the complex plastid require a signal peptide to direct the product to the endomembrane system and a transit peptide to direct the protein into the plastid, unlike plastid-targeted products in higher plants, which require only a transit peptide [7]. The model of complex plastid targeting [8] proposes that a signal sequence initially directs a nuclear encoded complex plastid protein to the endoplasmic reticulum (ER). Once in the ER, the signal sequence is cleaved, exposing the putative transit domain. The transit peptide then traffics the pre-protein to the plastid via the ER, golgi, and vesicular trafficking pathways. Upon entry into the complex plastid, the putative transit sequence is processed, producing the mature protein. However, previous studies have only used reporter

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genes tagged with apicoplast leaders to study this process. As these studies only identified the final location of the targeted reporter protein, they do not provide much insight into the pathway involved in the targeting process itself. The ribosomal small subunit 9 (rps9) in T. gondii is a nuclear encoded protein targeted to the apicoplast. The rps9 gene, absent in the apicoplast 35 kb genome, was recovered from polyA+ cDNA. Its genomic sequence harbors spliceosomal introns indicative of nuclear encoded genes and encodes a bipartite leader characteristic of nuclear encoded apicoplast proteins. Antibodies to rps9 detected the protein in the apicoplast by immunofluorescence assay [6]. We have employed deletion analyses to map the important domains involved in targeting nuclear encoded proteins to the apicoplast. Our results suggest that redundant signals exist within the leader sequence. We have also employed constructs with epitope tags inserted within the leader to explore the pathway involved in the targeting process. These results suggest that the targeting pathway does proceed through the ER as predicted by the model, and that processing of the leader is a multi-step process, with processing steps occurring both outside and at or within the apicoplast.

2. Materials and methods

2.1. Cell culture and parasite growth The RH strains with an HXGPRT knock-out mutant (NIH AIDS reference and Reagent Repository, Bethesda MD, http://www.niaid.nih.gov/reagent) were maintained by serial passage in human foreskin fibroblasts (HFF) or Vero cells. Host cells were grown in Dulbecco’s modified eagle medium (Gibco BRL) supplemented with 5% fetal bovine serum (FBS, Collaborative Biomedical Products).

2.2. Chemicals, antibodies, stains, enzymes Mycophenolic acids, xanthine, and 4%,6%-diamidino-2phenylindole (DAPI) were purchased from Sigma Chemical Corporation (St. Louis, MO). Rabbit antibodies to green fluorescence protein (GFP) were a gift from Pamela Silver [9]. Anti-GFP monoclonal antibodies and MitoTracker Red CMXRos were obtained from Molecular Probes (Eugene, OR). Antiserum to hemagglutinin tag (HA tag) was purchased from Roche (Indianapolis, IN). Antiserum to the ER chaperone BiP was kindly provided by Dr. Jay Bangs [10]. Restriction enzymes were purchased either from Promega (Madison, WI), or New England Biolabs (Beverly, MA).

2.3. GFP expression plasmids The parental pGRA-GFP plasmid [11] was created from GRA1/GFP5/GRA2-SK, which was kindly provided by Dr Kami Kim [12]. The plasmid pGFA-GFP contains the m-gfp5 gene from the thermostable folding mutant GFP5 [13]. The pGRA-GFP vector has the hxgprt minigene as a selectable marker [14]. The rps9 leader sequence, which includes the putative signal and transit sequence of rps9, was amplified from a clone with the full rps9 cDNA sequence using Taq polymerase (Promega) and 30 cycles of amplification with primers rps9.F1-Nsi1 (sense) and rps9.R156-Nsi1 (antisense) (Table 1). The resulting PCR product was ligated into the pCR2.1-TOPO vector (Invitrogen), transformed, and its sequence confirmed. The rps9(1– 156)-TOPO plasmid having the correct rps9 leader sequence was digested with NsiI and cloned into a NsiI, digested and phosphatase treated pGRA-GFP. A similar procedure was used to create all deletion constructs. Sequences of all primers are listed in Table 1.

2.4. Hemagluttinin tag expression plasmids For each construct, the HA tag was inserted at a different region of the putative rps9 transit sequence. The HA tag was constructed by annealing oligos HA.linker1 with HA.linker2 (Table 1) at 260 mM concentration in 50 mM NaCl, 10 mM Tris pH 8.0, 1 mM EDTA. The annealing reaction was heated to 94 °C for 2 min and gradually cooled to create a double strand HA linker with ends compatible with a ClaI and SpeI digested vector. A PCR-mediated, linker-scanning mutagenesis method [15] was used to create the HA tag constructs. The rps9(1–156) insert was ligated to an EcoR1 cut and phosphatase treated pGEM3Z vector (Promega). The resulting rps9(1–156)-GEM3Z plasmid which lacks SpeI and ClaI restriction sites was used as the template for PCR. Using outward directing primers with ClaI or SpeI sites and Pfu polymerase (Stratagene), linear PCR products with ClaI ad SpeI sites at each end were generated. Depending on the primers, ClaI and SpeI sites could be introduced anywhere along the length of the rps9 leader sequence. Table 1 lists all primer sequences used for these HA tagged constructs. Each amplified linear DNA was gel purified, digested with ClaI and SpeI, and ligated with the HA linker to produce plasmids having and HA tagged rps9 leader in pGEM3Z. Digestion of each plasmid with NsiI and ligation of each HA tag insert into a NsiI digested and phosphatase treated pGRA-GFP vector created plasmids HA38-GFP, HA55-GFP, HA84-GFP, HA109GFP, HA127-GFP, and HA143-GFP.

S. Yung et al. / Molecular & Biochemical Parasitology 118 (2001) 11–21 Table 1 PCR primers and linkers Name

Oligonucleotide sequencea

rps9.F1-NsiI

5%-ATGCATATGGCCCTCGAACG-TTGGT G-3% 5%-ATGCATGCC-TTCATAGTCCCTCAA -3% 5%-ATGCATGACT-ATGAAGGCAGTTCC -3% 5%-ATGCATGTTGCGTTGAGGGAC-3% 5%-ATGCATGAAATTCCTGGTAAAGCT -3% 5%-ATGCATAACCAAGGGAAGGACACGG A-3% 5%-ATGCATTGGGAGTGACCGAGGGGAG A-3% 5%-ATGCATATCTTCGAAAGGACATCC -3% 5%-ATGCATATCCGTCCCTAGAGCTGTG -3% 5%-ATGCATCGGCGTTCCAGGCTCAGG -3% 5%-ATGCATATAAGACCTTTTCCGGG-3% 5%-ATGCATCGTCAGCTTTCCAGAACCAG -3% 5%-ATGCATAAGATAGTCGGCTGCGTCTC -3% 5%-ATGCATGGCCTCCGCAATGATATCAA ATTC-3% 5%-ATGCATGATTGCTCCAGACTGCCCG3% 5%-ATGCATTTTGCTGTACTGTTCCTTC -3% 5%-CATCACCTTCACC-CTCTC-3% 5%-CGATTACCCATACGATGTTCCAGATT ACGCTA-3% 5%-CTAGTAGCGTAATCTGGAACATCGTA TGGGTAAT-3% 5%-GACTAGTCAACGCAACCTGCATAGCT T-3% 5%-CCATCGATA-GGGACTATGAAGGCAG TTCCG-3% 5%-GACTAGTGAAATTCCTGGTAAAGCTA TG-3% 5%-GACTAGTAAGCCGATTGGTTTGCAGG AGA-3% 5%-CCATCGATAACCAAGGGAAGGACAC GGA-3% 5%-GACTAGTGGGAGTGTCGGCCAGGTG AC-3% 5%-CGATCGATTGGGAGTGACCGAGGGG AGA-3% 5%-GACTAGTTCGCTTCCTAGTGACTGGG T-3% 5%-GGATCGATAGTTTCATGGACTGCATC TTC-3% 5%-GACTAGTGCGACACTCTCAAGCATTC -3% 5%-CCATCGATATCCGTCCCTAGAGCTGT G-3% 5%-GACTAGTAACAGCTTCTCATGGGGCA C-3% 5%-CCATCGATCGGCGTTCCAGGCTCAGG T-3%

rps9 transF34-NsiI rps9.R37-NsiI rps9.R41-NsiI rps9.R49-NsiI rps9.R55-NsiI rps9.R84-NsiI rps9.R104-NsiI rps9.R127-NsiI rps9.R143-NsiI rps9.R156-NsiI rps9.R169-NsiI rps9.R179-NsiI rps9.R208-NsiI rps9.R220-NsiI rps9.R270(full) -NsiI GFP.R2 primer HA.linker1 HA.linker2 rps9.38F-SpeI rps9.38R-ClaI rps9.49R-SpeI rps9.55F-SpeI rps9.55R-ClaI rps9.84F-SpeI rps9.84R-ClaI rps9.109F-SpeI rps9.109R-ClaI rps9.127F-SpeI rps9.127R-ClaI rps9.143F-SpeI rps9.143R-ClaI

a

Restriction endonuclease recognition sites are underlined.

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2.5. Electroporation and selection of stable transfectants HXGPRT deficient tachyzoites were harvested and resuspended (107 parasites total) in 300 ml of cytomix buffer (120 mM KCl, 0.15 mM CaCl2, 5 mM MgCl2, 2 mM EDTA, 10 mM KH2PO4, 25 mM HEPES-KOH pH 7.6, 2 mM ATP, 5 mM reduced glutathione) with 30–40 mg of sterilized plasmid DNA in a 2 mm gap cuvette (BioRad) and electroporated under previously described protocols [14]. After electroporation, parasites were left undisturbed for 15 min before inoclutating human foreskin fibroblasts (HFF) grown on glass coverslips (for fluorescent microscopy), T-25 cm2 flasks (for stable selection or for FACS), or 100× 20 mm tissue dishes with half confluent Vero cells (for cloning GFP positive plaques). Stable transfectants were selected using medium having 25 mg ml − 1 mycophenolic acid and 50 mg ml − 1 xanthine. For cloning directly from a 100×20 mm tissue plate, each plate was inoculated with 1 ml of the transfection solution. After overnight incubation, soft agar (0.9% w/v) was poured onto each plate [16]. Around 5–7 days later, plaques were screened under an inverted fluorescent microscope for GFP fluorescence. Sterile Pasteur pipette tips were used to pick positive plaques, which were inoculated into individual wells of a 24-well plate, or into a T-25 cm2 flask. Alternatively, cloning was accomplished by first inoculating 250 ml of transfection mixture into a T-25 cm2 flask. When parasites lysed the Vero cell monolayer, the polyclonal population was sterilely sorted for GFP fluorescence using a Becton Dickinson FACStarPlus to collect the brightest 2% of the parasites. Thirty thousand sorted positives were used to infect a 10 cm plate with a Vero cell monolayer for soft agar cloning. Most parasites expressing GFP fluorescence could be cloned and passaged stably for weeks without losing fluorescence intensity.

2.6. Florescence microscopy Intracellular parasites were grown in a monolayer of HFF adhered to coverslips. Extracellular parasites were resuspended in phosphate buffered saline (PBS) and settled onto poly-lysine coated coverslips. The coverslips were washed with PBS, fixed with 3% para-formaldehyde, permeablized with 0.5% Triton X-100, and incubated with the appropriate primary and secondary antibodies (1:1000 dilution). 4%,6%-diamidino-2-phenylindole (DAPI) was used as a DNA specific dye that stains the nucleus and apicoplast but not the mitochondria of T. gondii [4]. Between each change of reagent, the coverslips were washed three times with PBS. The coverslips were mounted with Mowiol onto glass slides and allowed to dry overnight before analysis.

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Immunofluorescence images were taken at the UAB imaging Facility with a Zeiss Axiovert microscope equipped with an ultraviolet filter set (Chroma Technology; Inc in Brattleboro, VT). Images were captured by a Photometrics Sensys cooled CCD, high resolution, monochromatic camera (Roper Scientific; Tucson, AZ). Image Acquisition Software was purchased from IPLab Spectrum from Scanalytics (Fairfax, VA).

2.7. Western blot analysis Parasites were lysed in SDS sample buffer and immediately boiled for 5 min. SDS gel electrophoresis and electroblotting onto immobilon membranes were performed according to standard protocols [17]. After blocking in 5% fat-free dry milk in TBS– Tween buffer (20 mM Tris– HCl pH 7.6, 137 mM NaCl, 0.1% w/v Tween 20), blots were incubated with anti-GFP polyclonal antibodies (1:3000 dilution) or anti-HA antibodies (1:3000 dilution). Bound antibodies were detected with the appropriate secondary antibodies conjugated to horseradish peroxidase. Supersignal West pico chemiluminescent substrate (Pierce) was used for detection according to the manufacturer’s protocol.

3. Results As a first step in mapping the domains involved in plastid targeting, a plasmid was constructed consisting of the full length rps9 leader sequence (designated rps9(1–156)) fused to a green fluorescent protein (GFP) reporter. The rps9(1– 156) parental plasmid was then used to prepare a series of nested deletions of the rps9 leader. Stable transfectants containing the full length construct and the deletions were examined to determine the location of the GFP reporter protein. Three different cellular localization patterns were found (Fig. 1). Some transfectants (e.g. rps9(1– 37)-GFP) displayed perinuclear labeling (Fig. 1A). This pattern of localization was identical to what was seen in parasites stained with antibodies to the endoplasmic reticulum (ER) chaperone BiP [18] (Fig. 1, panels B and C) suggesting that parasites exhibiting this pattern of fluorescence had the GFP reporter protein localized to the ER. In contrast, the constructs containing only the transit like domain of rps9 and lacking the putative ER entry signal sequence (amino acids 1– 34, e.g. rps9(34–156)GFP) displayed a different fluorescence pattern (Fig. 1, panels D–F). This pattern was identical to that seen in cells stained with the mitochondrial specific dye MitoTracker Red CMXRos [19], (Fig. 1, panels E and F), suggesting a mitochondrial location of the GFP. Finally, some of the constructs (e.g. the full length construct rps9(1–156)-GFP) produced a third pattern of fluorescence (Fig. 1, panels G– I) that co-localized with

the apicoplast marker, acyl carrier protein [6] (Fig. 1, panels H and I). Studies involving the various deletions in the leader are summarized in Fig. 2. As a first step in mapping the functional domains of the rps9 leader, deletions of the carboxyl terminus of rps9 were constructed to determine the minimal amount of the rps9 leader sufficient for apicoplast targeting. The construct rps9(1– 55)-GFP successfully targeted the GFP reporter protein to the apicoplast (Fig. 2). In contrast, a construct containing the first 49 amino acids of the leader (rps6(1–49)-GFP) displayed perinuclear staining with anti-GFP antibodies in an immunofluorescence assay (Fig. 2). These results suggest that the first 55 amino acids of the transit sequence are sufficient to successfully target the reporter to the apicoplast, and that at least part of the transit signal resides in the region encompassing amino acids 49– 55. As mentioned in Section 1, earlier studies have suggested that the apicoplast leader is bipartitate in nature, with the amino terminal end encoding an ER entry like signal and the remaining portion of the leader encoding a mitochondrial like transit signal. To further explore this hypothesis, a series of amino terminal deletions were constructed in the leader sequence. As predicted by the hypothesis proposing that the leader is bipartitate in nature, the N-terminus deletion plasmid rps9(34–156)-GFP, which lacked the putative signal sequence but encodes the remaining portion of the leader sequence exhibited mitochondrial fluorescence (Fig. 2). Similarly, a construct containing amino acids 34–55 (rps9(34–55)-GFP) was also found to target the GFP reporter protein to the mitochondrion (Fig. 2). In contrast, the reporter protein produced from construct rps9(34–49) GFP was localized to the cytoplasm. Together, these results suggest that amino acids 34–55 are sufficient for transit targeting, and that at least part of the information essential for targeting resides in amino acids 49–55. Both the carboxyl and amino terminal deletion studies suggested that amino acids 49–55 were important in targeting the GFP reporter to the apicoplast. In order to determine if these residues were essential for the targeting process, a plasmid was constructed consisting of the intact leader sequence with amino acids 49–55 deleted. Surprisingly, this construct (rps9(D49–55)GFP targeted the encoded GFP reporter protein to the apicoplast (Fig. 2). This result suggests that some redundancy might exist in the targeting information encoded by the rps9 leader. The deletion experiments described above suggested that the first 55 amino acids of the leader are sufficient to correctly target the GFP reporter protein to the apicoplast. However, the deletion studies resulted in leaders that were severely modified relative to the full length leader sequence. Furthermore, the deletion stud-

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Fig. 1. Localization of GFP in constructs containing deletions of the rps9 leader. Panels (A – C) localization of GFP in cells transfected with rps9(1 – 37)-GFP. Panel (A) Reactivity of anti-GFP antibodies in transfected cells. Panel (B) Reactivity of antibodies of the ER marker protein BIP in transfected cells. Panel (C) Merged image of panels A and B. Panels (D – F) Localization of GFP in cells transfected with rps9(34 –156)GFP. Panel (D) Reactivity of anti-GFP antibodies in transfected cells. Panel (E) Cells stained with the mitochondrial specific dye MitoTracker Red CMXRos. Panel (F) Merged image of panels D and E. Panels (G – I) Localization of GFP in cells transfected with rps9(1 – 156)-GFP. Panel (G) Reactivity of anti-GFP antibodies in transfected cells. Panel (H) Reactivity of antibodies to the apicoplast protein acyl carrier protein. Panel (I) Merged image of panels G and H.

ies only provided information as to the localization of the reporter protein and did not shed any light on the steps involved in processing the leader itself. To address these questions, linker scanner mutagenesis was used to insert a hemagglutinin (HA) epitope tag at different locations in the transit domain of the rps9 leader. The insertion sites of HA tags within the transit domain are shown schematically in Fig. 3. Constructs were numbered ac-

cording to the insertional site. For example, HA38-GFP has the HA tag inserted after amino acid 38 of the rps9 leader. Stable lines containing the HA tagged rps9-GFP constructs were obtained for all of the tagged constructs with the exception of HA55-GFP. No stable transfectants containing this construct were obtained after numerous attempts. Therefore, studies of this construct were carried out on transiently transfected lines.

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A summary of the results obtained in the localization studies involving the HA tagged constructs is provided in Fig. 3, and examples of the immunolocalization patterns obtained are shown in Fig. 4. Since the HA tag is relatively small (13 amino acids), we predicted that targeting of the HA tagged constructs would less disrupted than in the deletion constructs described above. In support of this hypothesis, HA tagging did not totally disrupt targeting in any of the tagged constructs, since GFP fluorescence was seen at the apicoplast in all of the insertional mutants (Figs. 3 and 4). However, targeting was somewhat affected in these parasites, as GFP was also detectable in the perinuclear space of constructs tagged in the amino terminal half of the leader (HA38-GFP and HA55-GFP) (Figs. 3 and 4). Thus, although these constructs were able to target the reporter to its correct location, this process was not as efficient as with the parental leader construct, where GFP was only detectable in the apicoplast (e.g. Fig. 1, panel C). In contrast to the results obtained with parasites tagged in the amino terminal portion of the leader, the three constructs tagged in the carboxyl terminal portion of the leader only contained GFP in

the apicoplast (Figs. 3 and 4). This suggested that processing in these lines was not affected by the incorporation of the HA tag. The presence of the HA tag in the leader of the tagged lines made it possible to localize the leader sequence in the transfected parasites, and gain some insight into the processing pathway involved in maturation of apicoplast targeted nuclear encoded proteins. As discussed above, constructs containing the HA tag inserted in the amino terminal half of the transit sequence (HA-38-GFP and HA55-GFP) all contained GFP in both the perinuclear space and in the apicoplast. However, when lines containing these constructs were probed with the anti HA tag antibody, protein was only detected in the perinuclear space (Figs. 3 and 4). In contrast, the three constructs containing the HA tag inserted in the carboxyl terminal half of the leader (HA109-GFP, HA127-GFP and HA148-GFP) exhibited a different localization pattern. In these lines, the protein that reacted with the anti-HA tag antibody localized only to the apicoplast (Figs. 3 and 4). These results, when taken together, suggested that the first 55 amino acids of the leader sequence are removed prior

Fig. 2. Summary of targeting of rps9-GFP deletion constructs. The boundaries of the deletion constructs are schematically indicated. The deletion constructs contained varying portions of the signal sequence (white boxes) the transit sequence (grey boxes) and the mature rps9 sequence (black boxes). All of the deletion constructs contained the complete GFP open reading frame (Hatched box). The location of the GFP protein is given to the right of each deletion diagram. P, perinuclear; A, apicoplast; M, mitochondria; C, cytoplasm.

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Fig. 3. Localization of GFP and the HA tag in parasites transfected with HA tagged leaders. HA tagged constructs were generated by linker mediated insertion into rps9(1 –156) as described in Section 2. The white boxes indicate the putative signal sequence of rps9. Gray boxes represent the putative transit sequence. Black boxes represent the mature portion of rps9. In each panel, ex, extracellular; in, intracellular; P, perinuclear; A, apicoplast localization.

to or during import of the protein into the apicoplast, while the carboxyl terminal domain of the leader (from amino acids 109– 158) is retained upon import. To further investigate the pathway involved in processing of the leader, western blots were prepared with extracts derived from two HA tagged constructs representing the two different patterns of HA localization shown in Figs. 3 and 4. The blots were developed with antibodies to GFP and with antibodies recognizing the HA tag. Anti-GFP antibodies detected three proteins with molecular weights of 47, 41 and 39 kDa in cells transfected with HA38-GFP (Fig. 5, panel A). In cells transfected with HA109-GFP, bands of 47 and 41 kDa were detected. In parasites transfected with the untagged rps9(1–156)-GFP construct, bands of 46 and 39 kDa were detected. The 46 kDa band is approximately 1 kDa smaller than the largest band detected in the two tagged constructs. This difference roughly corresponds to the predicted increase in mass that would result from the addition of the HA tag to the leader in the tagged constructs (1.4 kDa). In the Western blot developed using the anti HA tag antibody, a different pattern was found (Fig. 5, panel B). Here, the anti-HA tag antibody recognized the 47 kDa band but not the 39 kDa band in parasites transfected with HA38-GFP. In parasites transfected with HA109-GFP, two bands of 47 and 40 kDa were recognized, identical to the pattern seen in the western blot

developed with the anti-GFP antibody. As expected, transfectants of the untagged leader construct rps9(1– 156)-GFP did not react with anti-HA antibodies (Fig. 5, panel B).

4. Discussion As described in Section 1, it has been hypothesized that the leader of apicoplast targeted proteins is bipartite in nature. Nuclear encoded apicoplast proteins are thought to first be directed to enter the secretory pathway via a signal sequence present at the amino terminal end of the peptide. Once directed to the secretory pathway, the signal sequence is cleaved, exposing a second domain. This second domain directs the protein to the apicoplast. After import into the apicoplast, the transit-like domain is removed, resulting in the production of the mature protein. This model is supported by previous studies employing reporter proteins tagged with leader sequences derived from T. gondii for acyl carrier protein [6] and rps9 [20]. However, because these studies relied on detection of the reporter proteins alone, they were unable to detect the intermediates in the targeting pathway. In the current study, we have employed deletion and HA tagged constructs of the rps9 leader attached to the amino terminal end of a GFP reporter. The deletion constructs allowed us to

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map the domains important in the targeting process, while the HA tagged constructs have allowed us to follow intermediates in the targeting pathway, shedding light on the steps involved in targeting and in processing the leader sequence. Data derived from studies of the carboxyl terminal deletions demonstrated that large C-terminal portions of the rps9 leader could be deleted without affecting apicoplast targeting of GFP. These results suggested that the carboxyl terminal 102 amino acids of the leader were not necessary for correct targeting. Furthermore, the first 55 amino acids of the rps9 leader were found to be sufficient for apicoplast targeting. In contrast, the first 49 amino acids were not sufficient to target the reporter to the apicoplast. These results suggested that amino acids 49–55 contain important information for the correct targeting to the apicoplast. However, additional studies suggest that some redundancy may exist in signals encoded in the leader. For example, we noted that while constructs containing only the first 55 amino acids of the rps9 leader were capable of targeting the GFP reporter to the apicoplast, these transfectants did not display as strong an in vivo GFP fluorescence as did transfectants containing constructs with the full length leader (data not shown). This suggests that while the first 55 amino acids were capable of targeting the

reporter to the apicoplast that this process might not be as efficient as it was with constructs containing the full length leader. Furthermore, although the nested deletion studies suggested that amino acids 49–55 contained information essential to direct the targeting to the apicoplast, a construct deleting only amino acids 49–55 was targeted to the apicoplast. These results, when taken together, suggest that while the first 55 amino acids are sufficient to target a reporter to the apicoplast, some redundancy exists in the targeting signal encoded in the leader. This finding is in line with previous studies that have demonstrated that plant plastid and mitochondrial targeting signals generally contain redundant information and that small deletions or insertions, therefore, do not generally alter targeting [21,22]. A positively charged amphiphilic alpha helix structure could be predicted between amino acids 40– 52 of the rps9 leader, and the PSORT [23] and TargetP [24] programs predicted the rps9 putative transit-like domain to be a mitochondrial targeting peptide. These observations may explain the mitochondrial targeting ability of the transit domain of rps9 when expressed in the absence of the putative signal sequence encoded in amino acids 1–34. This finding suggests that the apicoplast transit signal of rps9 may have much in common with the mitochondrial transit signal.

Fig. 4. Immunolocalization assays of GFP and HA tag sequence in hemagglutinin tag insertion transfectants. Arrows point to the apicoplasts. Not all apicoplasts were in the photographed focal plane.

S. Yung et al. / Molecular & Biochemical Parasitology 118 (2001) 11–21

Fig. 5. Western blot analysis of HA tagged constructs. Extracts of untagged (rps9 (1 – 156)) and HA tagged extracellular parasites were used to prepare duplicate western blots which were probed with anti-GFP and anti-HA tag antibodies. HA tagged constructs were generated by linker mediated insertion into rps9(1 –156) as described in Section 2. Panel (A) Western blot probed with anti-GFP antibodies. Panel (B) Western blot probed with anti-HA tag antibodies.

As mentioned above, small insertions in the leader sequence do not severely affect targeting in mitochondria and plant plastids [21,22]. This also seems to be the case in apicoplast targeting in T. gondii, as all of the HA tagged constructs appeared to target the GFP reporter to the apicoplast. However, some effects on targeting were seen in constructs containing the HA tags. For example, the HA38-GFP and HA55-GFP constructs contained GFP protein both in the apicoplast and in the perinuclear region. The GFP in the perinuclear region co-localized with anti-BiP antibodies (data not shown). Since the perinuclear nuclear envelope has been proposed as the intermediary between the ER and Golgi [25], this result supports the model in which the nuclear encoded complex plastid protein first transverses the secretory system. Furthermore, like rps9(1–55)-GFP, the fluorescence intensity of HA38GFP was noticeably dimmer than that seen in the untagged full length construct (data not shown). These results suggest that the amino terminal HA tagged constructs were targeted correctly, but that the targeting process was less efficient than in constructs containing the unmutated leader. As a result, precursors may have either been backed up in the secretory system, requiring more time to be imported in these mutants, or some proportion of the products may be mistargeted. In contrast to the amino terminal tagged constructs, transfectants with the HA tag inserted in the carboxyl terminal half of the leader contained all of the GFP reporter protein in the apicoplast. Having the HA tag

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near the carboxyl terminus of the transit domain, therefore, did not appear to affect apicoplast targeting. Western blot and immunolocalization studies utilizing anti-GFP and anti-HA tag antibodies suggest that processing of the rps9 leader is a multi-step process. In parasites transfected with HA38-GFP and HA-55-GFP, GFP was localized to both the perinuclear space and the apicoplast, while the HA tag was present only in the perinuclear space. Furthermore, in western blot analysis of these constructs only the largest protein recognized by the GFP antibodies was recognized by the HA tag antibodies. In contrast, in parasites transfected with the HA109-GFP, HA tagged protein was located in the apicoplast, and the peptides recognized by both the GFP and HA tag antibodies were identical. These results, when taken together, suggest that some sort of cleavage event is occurring between amino acids 55 and 109 of the leader, as HA tags inserted at or before position 55 are not imported into the apicoplast, while HA tags inserted at position 109 are imported into the apicoplast. The 39 kDa peptide common to all the constructs cannot represent a fully processed product, as it is 9 kDa larger than mature GFP expressed without a leader. It is possible that the recognition site for the final cleavage event resulting in the removal of the last portion of the rps9 leader is encoded in the mature rps9 protein itself. If this was the case, this site would be absent in the GFP chimeras, and the final cleavage event would not occur. However, western blots detect the 39 kDa peptide in rps9(1–169) GFP transfected parasites. Nothing of the size predicted for cleavage at the amino terminal end of the mature rps9 protein is seen (data not shown). Since rps9(1–169) GFP contains roughly the first 25 amino acids of the mature rps9 protein, this result suggests that if a signal for the final cleavage event is encoded in rps9, it is not localized within the first 25 amino acids of the mature protein. It is possible that, as has been documented in the case of ribosomal protein L-18 of Chlamydomonas reinharditii [26], final processing may occur during or after ribosomal assembly. Further work will be needed to address this possibility. These results, when taken together, suggest a model for the pathway involved in the trafficking of the rps9 leader tagged GFP constructs (Fig. 6). The peptide containing the full length bipartitate leader is co-translationally introduced into the ER. At this point the signal sequence (roughly amino acids 1–34) is removed, exposing the transit sequence. The peptide is then translocated to the apicoplast through the perinuclear space. Trafficking to the apicoplast involves a recognition site encoded in amino acids 49–55 of the leader sequence, as well as a redundant site or sites encoded elsewhere in the leader. During the trafficking process, the transit peptide is cleaved between amino acids 55

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Fig. 6. Proposed model for rps9 processing. Details of the model are given in the text. The proposed steps in the model are as follows. Step 1, cleavage and removal of the signal sequence (ca. amino acids 1 – 34). Steps 2 and 3, cleavage of the leader sequence at a site between amino acids 55 and 109. Step 4, import of the protein into the apicoplast and removal of the remainder of the leader. The bracket surrounding steps 2 and 3 indicates that the cellular compartment where the cleavage of the leader sequence at the site between amino acids 55 and 109 occurs is not known.

and 109. The 41 kDa peptide detected by the anti-GFP antibodies in extracts prepared from HA38-GFP parasites may represent an intermediate in this process, or may represent an aberrantly processed version of the peptide derived from mistargeted proteins, as discussed above. Finally, once in the apicoplast, the remaining portion of the leader is removed, resulting in the production of the mature protein. This final processing step may occur concurrently with ribosome assembly. In summary, the data reported above demonstrate that the amino terminal 55 amino acids of the rps9 leader are sufficient to target a reporter gene to the apicoplast of T. gondii, but that other sites encoded in the leader may also be involved in this process. Experiments employing the HA tagged versions of the leader demonstrate that, as hypothesized, trafficking of the nuclear encoded apicoplast proteins proceeds through the ER. Finally, these data suggest that processing of the HA tagged leaders is a multi-step process, involving both signal sequence removal and a cleavage event

between amino acids 55 and 109 of the leader. Further experiments will be necessary to map the redundant targeting signals in the rps9 leader, to demonstrate the precursor product relationship between the 47 kDa product and the 39 kDa product, to determine exactly where the cleavage event that gives rise to the 39 kDa product occurs, and to study the final cleavage events that result in the production of fully mature RPS9 protein.

Acknowledgements The authors thank Con Beckers and members of the Beckers, Lang–Unnasch, and Unnasch labs for helpful suggestions and discussions. We thank the technical help of Albert Tousson and Marion Spell for microscopy and FACS. This work was supported by a grant from the US National Institutes of Health (RO1 Al 48737).

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