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J. Biol. Chem., Vol. 277, Issue 43, 41240-41246, October 25, 2002

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Proteolytic Processing of TgIMC1 during Maturation of the Membrane Skeleton of Toxoplasma gondii^*

Tara Mann, Elizabeth Gaskins^§, and Con Beckers^§¶

From the ^§ Division of Geographic Medicine and the  Department of Cell Biology, University of Alabama at Birmingham, Birmingham, Alabama 35294-2170

Received for publication, May 22, 2002, and in revised form, July 26, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Membrane skeletons play an important role in the maintenance of cell shape and integrity in many cell types. In the protozoan parasite Toxoplasma gondii this function is performed by the subpellicular network, a resilient structure composed of tightly interwoven 10-nm filaments. We report here that this network is assembled at an early stage in the development of daughter parasites. The networks of immature and mature parasites differ dramatically with respect to their stability. Although in immature parasites the network is completely solubilized by detergent, the network in mature parasites is entirely detergent-resistant. Conversion of the detergent-labile to the detergent-resistant network occurs late in daughter cell development and appears to be coupled to proteolytic processing of the carboxyl terminus of TgIMC1, the major subunit of the network filaments. A single cysteine residue in the TgIMC1 carboxyl terminus was found to be essential for this processing event. The dramatic change in resistance to detergent extraction probably reflects an overall change in structural stability of the subpellicular network that accompanies maturation of daughter parasites and allows a switch from an assembly-competent but loose structure to one that is rigid and offers mechanical strength to the mature parasite.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Membrane skeletons, composed of the plasma membrane and its organized underlying coat, are found in many cell types and are essential for mechanical strength and the maintenance of cell shape. Two widely divergent examples are the spectrin-based membrane skeletal system of the erythrocyte and the articulin-based system in the unicellular Euglena. In the erythrocyte, spectrin along with its associated proteins such as ankyrin form a meshwork underlying the plasma membrane (1). In the Euglena, two articulin proteins interact stoichiometrically to form a membrane skeletal system consisting of 40 interdigitating strips (2-4). Along with the articulins of the Euglena, a number of unique cytoskeletal systems have been described in protists, including the giardins (5, 6), assemblins (7), and tetrins (8) as well as the components of the subpellicular network, a novel cytoskeletal structure recently identified in the protozoan parasite Toxoplasma gondii (9).

T. gondii is an obligate intracellular parasite that infects a wide range of nucleated cells. Human infection with the parasite is generally asymptomatic except in patients with compromised immune systems and during the early stages of pregnancy (10, 11). Like the other members of the phylum Apicomplexa, such as Plasmodium sp. or Cryptosporidium, Toxoplasma has a number of prominent structural elements. Chief among these is the pellicle, composed of the plasma membrane and the inner membrane complex, flattened membrane cisternae possibly derived from the endoplasmic reticulum (12, 13). Recently, we described a filamentous network, the subpellicular network, that is associated with the cytoplasmic face of the inner membrane complex along the entire length of the parasite. This resilient structure appears to act as the membrane skeleton of the pellicle and thereby lends the parasite the mechanical strength it needs for survival. The subpellicular network is composed of a fine mesh of 10-nm filaments and appears to consist, at least in large part, of two novel proteins, TgIMC1 and TgIMC2, that have some structural similarity to intermediate filaments in animal cells (9). In addition to the subpellicular network, Toxoplasma also possesses a tubulin-based cytoskeleton that lies on the cytoplasmic face of the subpellicular network and consists of 22 subpellicular microtubules, a conoid, and polar ring (14-16).

After invasion of the host cell, the parasite establishes itself inside a vacuole of its own making, the parasitophorous vacuole, and initiates replication. In Toxoplasma, cell division occurs through a specialized form of binary fission, endodyogeny, in which two complete daughter cells are formed within the mother cell. This process begins with the formation of two new conoids and polar rings in close apposition to the nucleus. This is followed by the gradual elongation of the subpellicular microtubules of the daughter cells and acquisition of an inner membrane complex. After nuclear division, the daughter nuclei, along with other organelles, are partitioned between the developing daughter parasites. These continue to enlarge, and as they fill the mother cell, the remaining maternal organelles as well as the inner membrane complex degenerate. At this time, the maternal plasma membrane associates with the inner membrane complex of the daughter cells, generating two parasites with complete pellicles. This unusual mode of replication ensures that the daughter parasites are contained and protected within a fully infectious mother cell until replication is completed (17-19).

We describe here that the subpellicular network of daughter parasites begins to assemble during an early stage in parasite replication. In addition, we have determined that the subpellicular networks in mother and daughter parasites differ markedly in their stability. Although the network in mother parasites is essentially resistant to extraction with detergent, the same structure in daughter parasites is highly susceptible. The different stabilities may reflect the need of the daughter cell network to remain plastic during growth and the need for the mother cell network to provide mechanical strength. The conversion from a detergent-sensitive to a detergent-resistant network appears to be coupled to the carboxyl-terminal proteolytic processing of TgIMC1. These data suggest that T. gondii has developed a unique process to control the assembly and mechanical strength of its membrane skeleton during cell division. This appears specifically designed to allow for the generation of infectious daughter parasites without exposing them to their hostile environment before their membrane skeleton is fully formed and stable.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials-- All chemical reagents were obtained from Fisher Scientific (Pittsburgh, PA) unless otherwise noted. Pfu Turbo was purchased from Stratagene (La Jolla, CA). All restriction enzymes and T4 DNA ligase were obtained from New England BioLabs (Beverly, MA). All oligonucleotides were purchased from Qiagen (Alameda, CA).

Culture of Parasites-- The RH (HXGPRT) strain of T. gondii (kindly provided by Dr. David Roos, University of Pennsylvania) was maintained by serial passage in confluent cultures of human foreskin fibroblasts (HFF)¹ or VERO cells in -minimal essential medium (Mediatech, Inc., Herndon, VA) supplemented with 10% fetal bovine serum (HyClone, Logan, UT), 2 mM glutamine, 100 IU/ml penicillin, and 100 µg/ml streptomycin. Parasites were harvested by syringe passage followed by centrifugation at 2000 rpm for 5 min.

Construction of Toxoplasma Expression Vectors-- For the construction of the expression vector pEXP, the 5'- and 3'-untranslated regions of SAG1 were cloned into pBluescript KS. The SAG1 open reading frame was replaced by a short linker region containing an NdeI site overlapping the original start codon of SAG1 and an NotI site. The HXGPRT selection marker under control of its own promoter was inserted downstream of the SAG1 3'-untranslated region.

For the production of vectors containing either an amino-terminal or carboxyl-terminal HA tag, pEXP1 was digested with NdeI and NotI, and linkers encoding the HA tag were inserted between these sites. To create amino-terminally tagged proteins the vector pEXP-NtermHA was created using the oligonucleotides 5'-tatgtacccatacgatgttcctgactatgcgggcggatccctcagc-3' and 5'-ggccgctgagggatccgcccgcatgtcaggaacatcgtagggtaca-3'. In this vector unique BamHI site (underlined) was introduced immediately following the HA tag to allow insertion of the sequence to be tagged.

To create carboxyl-terminally tagged proteins, the vector pEXP-CtermHA was generated using the oligonucleotides 5'-tatgctcagtatacccatacgatgttcctgactatgcgggctaagc-3' and 5'-ggcgcttagcccgcatagtcaggaacatcgtatgggtatactgagca-3'. In this vector a unique Bst1107 (underlined) was introduced immediately preceding the HA tag to allow insertion of the sequence to be tagged. All constructs were confirmed by sequencing (Keck Biotechnology Resource Laboratory, Yale University, New Haven, CT).

HA-tagged TgIMC1-- For the production of TgIMC1 with either an amino- or carboxyl-terminal HA tag, the TgIMC1 open reading frame was amplified by PCR from a full-length cDNA clone and cloned into pEXP-NtermHA or pEXP-CtermHA. To introduce an amino-terminal HA tag into IMC1, PCR was performed using the primer 5'-ggatccatgtttaaggactgcgccgat-3', which introduces a unique BamHI site (underlined) at the start of the TgIMC1 open reading frame, and 5'-gcggccgcttagcactggcatcggcac-3', which introduces a NotI site immediately after the TgIMC1 stop codon. To introduce a carboxyl-terminal HA tag, PCR was performed using 5'-gacctccatatgtttaaggactgcgccg-3', which introduces an NdeI site (underlined) at the start codon of TgIMC1, and 5'-cgaggtatacgcactggcatcggcacac-3', which introduces a Bst1107 site immediately preceding the TgIMC1 stop codon. The resulting constructs were named pHA-IMC1 and pIMC1-HA.

TgIMC1 Carboxyl-terminal Deletion Mutants-- Full-length TgIMC1 cloned, as described above, in the pEXP-CtermHA vector was used as the PCR template. An amino-terminal primer that binds in the SAG1 5'-untranslated region, 5'-cacacggttgtatgtcgg-3', was used with the following carboxyl-terminal primers containing a Bst1107 site (underlined) after the last desired amino acid: IMC1(592-609)-5'-gaggtatacctgggcagcttccggttctggcgctgc-3'; IMC1(595-609)-5'-ctactagtatacgacgcagcactgggcagcttccgg-3'; IMC1(598-609)-5'-ctactagtataccgcgcacatgacgcagcac-3'; IMC1(601-609)-5'-ctactagtatacgccgcagaccgcgcacatgacg-3'; IMC1(605-609)-5'-ctactagtatacaccatcaccgccgcagaccgc-3'. PCR products were gel-purified and inserted between the NdeI and Bst1107 sites of pEXP-CtermHA.

TgIMC1 Carboxyl-terminal Point Mutants-- The last 333 nucleotide acids of the TgIMC1 open reading frame along with the HA tag were moved from pIMC1-HA and cloned into pBluescript SKII using the restriction sites XmaI and NotI. All point mutations of interest were generated by inverse PCR using this plasmid as template. The mutagenic primers IMC1(C592T)-5'-gcacatgacgcaggtctgggcagcttcc-3', IMC1(C593T)-5'-gcacatgacggtgcactgggcagcttcc-3', IMC1(V594G)-5'-gcacatgccgcagcactgggcagcttc-3', IMC1(M595G)-5'-gcacccgacgcagcactgggcagcttcc-3' were used with the primer 5'-gcagtctgcggcggtgatggtgtg-3'. The mutagenic primers IMC1(C596T)-5'-accgcggtctgcggcggtgatg-3', IMC1(A597G)-5'-tgcggggtctgcggcggtgatg-3' and IMC1(V598G)-5'-tgcgcgggctgcggcggtgatg-3' were used with the primer 5'-catgacgcagcactgggcagcttcc-3'. The resulting PCR products were gel-purified, recircularized with T4 DNA ligase, and transformed in to Escherichia coli JM109. The mutagenized region of IMC1 along with the HA tag were excised using BsrGI and NotI and inserted between the BsrGI and NotI sites of pIMC1-HA.

TgIMC1-(1-586)-YFP Fusion Expression-- For in situ extraction experiments, a sequence containing residues 1-586 of IMC1 was amplified by PCR from the plasmid ptub-IMC1-YFP/sagCAT (20) using a forward primer that binds in the tubulin promoter region, 5'-cgcgcagaagacatccaccaaacg-3' and a reverse primer, 5'-gtccctaggaactggcttgatcacgtc-3', which introduces an AvrII site (underlined) after residue 586. The PCR product was gel-purified and digested with BglII and AvrII for cloning into the similar digested ptub-IMC1-YFP/sagCAT.

Transfection of Parasites-- Extracellular parasites were transfected with 50 µg of circular plasmid DNA by electroporation in a 2-mm cuvette at 1.5 kV, 25 microfarads using a Bio-Rad Gene Pulser II (Bio-Rad Laboratories). The parasites were then added to a confluent monolayer of HFF cells and allowed to invade for 4-5 h. When transfected with derivatives of pEXP, they were subjected to selection using 50 µg/ml xanthine (Sigma Chemical Co., St. Louis, MO) and 25 µg/ml mycophenolic acid (Roche Molecular Biochemicals, Indianapolis, IN).

Toxoplasma Protein Turnover-- A confluent monolayer of HFF cells in a T75 flask was infected with 3 × 10⁷ parasites. The parasites were allowed to invade host cells and replicate for 16-18 h. The normal growth medium was removed, and the parasite-infected cells were incubated 1 h in methionine- and cysteine-free medium, followed by the addition of 0.7 mCi of [³⁵S]methionine/cysteine (Amersham Biosciences, Piscataway, NJ). The incubation was continued for 20-24 h after which the parasites were harvested as usual and washed in normal growth medium containing methionine and cysteine.

For each time point one T25 flask with confluent HFF cells was infected with 10⁷ radiolabeled parasites, and coverslips of HFF cells were infected with 10⁵ parasites each. After a 2-h invasion period, all culture supernatants were aspirated and replaced with fresh medium. At the indicated times, a set of infected coverslips was fixed for 5 min in cold methanol and stored at 20 °C, and parasites were harvested from a T25 flask in cold PBS, pelleted, and stored at 20 °C. After collection of the last time point, sequential immunoprecipitations were performed on the sample from each time point using anti-TgIMC1 and anti-TgCDPK1 antisera. Immunoprecipitates were analyzed by SDS-PAGE and fluorography. Densitometric analysis of film was performed using Scion Image (Scion Corp., Frederick, MD). The extent of parasite replication was determined by counting the average number of parasites per vacuole by phase contrast microscopy (n = 2). For the sample treated with pyrimethamine, the 2-h invasion period was followed by the addition of fresh media containing 1 µM pyrimethamine (Sigma).

Pulse-chase Analysis of TgIMC1 Processing-- Confluent monolayers of HFF cells in two T25 flasks were infected with 10⁷ parasites for 16-18 h. The normal growth medium was removed, and the parasite-infected cells were incubated 1 h in methionine- and cysteine-free medium, followed by the addition of 0.7 mCi of [³⁵S]methionine/cysteine (Amersham Biosciences). After a 30-min incubation, one flask was placed on ice. The labeling medium in the second flask was replaced with normal growth medium containing 1 mM methionine and incubated for an additional 4 h at 37 °C. Infected cells were harvested on ice from both flasks by scraping and pelleted by centrifugation for 5 min at 2000 rpm. TgIMC1 was immunoprecipitated from both samples as described below.

Immunoprecipitation-- Parasite pellets were resuspended in 100 µl of 1% SDS and subjected to three rounds of freezing and heating for 5 min at 95 °C. 900 µl of 2% Triton X-100 in TBS and protease inhibitors were added followed by a 5-min incubation on ice. The lysates were centrifuged for 15 min at 14,000 rpm. The supernatant was incubated with primary antibody for 1 h on ice followed by the addition of protein A-Sepharose (Zymed Laboratories Inc., San Francisco, CA) for another hour at 4 °C. Immune complexes were pelleted and washed three times in 1% Triton X-100 in PBS. The immunoprecipitated proteins were released by resuspension in SDS sample buffer and separated by SDS-PAGE. For radiolabeled samples, electrophoresis gels were treated with 0.125 M sodium salicylate, dried, and exposed to x-ray film.

Detergent Extraction of TgIMC1-- HFF cells grown in 25-cm² flasks were infected with 10⁷ untransfected Toxoplasma or parasites expressing full-length IMC1 with a carboxyl-terminal HA tag. After 16-20 h, the HFF cells were washed twice with cold TBS and scraped into 2 ml of TBS. Parasites were released by passage through a 25-gauge needle and collected by centrifugation for 5 min at 2,000 rpm. Parasites were solubilized in 1% DOC in TBS for 5 min on ice in the presence of protease inhibitors. The DOC-soluble and insoluble materials were prepared by centrifugation for 10 min at 14,000 rpm. The DOC-insoluble pellet was again extracted with DOC as above, and the DOC-soluble fractions were pooled. Fractions corresponding to 10⁶ parasites were analyzed by SDS-PAGE on a 6% gel and immunoblot with anti-TgIMC1 or anti-HA antibodies.

SDS-PAGE and Immunoblotting-- Protein preparations were separated by SDS-PAGE on 12% polyacrylamide gels as described previously (21). Where indicated, proteins were transferred to nitrocellulose and probed with different antisera as described before (22). Bound antibodies were detected using the Super Signal kit from (Pierce, Rockford, IL).

Immunofluorescence-- For immunofluorescence on infected parasites, coverslips of confluent or semi-confluent HFF cells were infected with 10⁵ parasites. Parasites were allowed to replicate overnight before being fixed for 5 min in cold methanol. Extracellular parasites washed in PBS were allowed to adhere to poly-L-lysine coated glass coverslips for 15 min at room temperature and subsequently washed in PBS to remove unbound parasites. Attached parasites were extracted 5 min in 1% DOC in PBS, washed three times in PBS, and fixed for 5 min in cold methanol or first extracted with 1% DOC followed by methanol fixation. Samples were incubated for 30 min in primary antibodies diluted 1:500 (HA) or 1:1000 (anti-IMC1) in 3% bovine serum albumin in PBS. Bound antibodies were visualized using fluorescein or rhodamine-conjugated secondary antibodies at the dilutions suggested by the manufacturer (Bio-Rad Laboratories, Hercules, CA). The coverslips were mounted in MOWIOL. Epifluorescence microscopy was performed using an Olympus BX60, and images were photographed using Kodak T-max 400 film. Individual negatives were scanned using a Nikon LS-2000 scanner (Nikon Corp., Melville, NY), and images were processed in Adobe Photoshop (Adobe Photosystems, Inc., San Jose, CA).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Dynamics of the Subpellicular Network of Toxoplasma during Replication-- To understand the assembly properties of the subpellicular network, we examined the formation of the subpellicular network during endodyogeny. As can be seen in Fig. 1, the network of the new daughter cells can first be detected at a very early stage of endodyogeny, prior to any evidence of nuclear division. The mother cell network does not appear to change during assembly of the daughter cell network. Only during the final stages of endodyogeny is there any evidence for disruption of the network in the mother cell, as judged by a reduction in the fluorescence signal in that structure. The proteins that make up the subpellicular network of the mother cell do not appear to be reused during assembly of the network in the daughter cells. TgCDPK1, a cytoplasmic protein kinase, displays a half-life of 32 h over a period encompassing several 8-h Toxoplasma life cycles and is therefore likely reused (Fig. 2). The TgIMC1 in newly invaded parasites, on the other hand, appears to be relatively stable during the first 12 h of intracellular development but is degraded rapidly at the time the first parasite cell divisions become evident (Fig. 2). This result indicates that the proteins that make up the subpellicular network are not recycled and reused for the assembly of the daughter cells but are instead degraded during the late stages of endodyogeny. This conclusion is strengthened by the observation that the inhibition of parasite replication by pyrimethamine prevents TgIMC1 degradation almost completely (Fig. 2).

View larger version (40K): [in this window] [in a new window]   Fig. 1.   TgIMC1 localization during parasite replication. Examination of replicating parasites with anti-IMC1 antibodies reveals labeling of the mother cell until the time of budding as well as labeling of daughter cells throughout endodyogeny beginning at the very early stages of replication. A-F are representative of anti-TgIMC1 localization as replication proceeds.

View larger version (26K): [in this window] [in a new window]   Fig. 2.   TgIMC1 is degraded rather than recycled during Toxoplasma cell division. A, the turnover rate of TgIMC1 and its relationship to Toxoplasma replication was determined by infecting HFF monolayers with metabolically labeled parasites as described under "Experimental Procedures." After removal of non-invaded parasites at 2 h, samples were taken approximately every 12 h over a period of 40 h. The average number of parasites per vacuole at each time point was determined to follow parasite replication. Immunoprecipitations were performed using anti-TgIMC1 antibodies and anti-TgCDPK1 antibodies. Densitometric analysis was performed, and the data were graphed as a fraction of the optical density observed at the 2-h time point. B, to verify that TgIMC degradation is linked to Toxoplasma replication, TgIMC1 was immunoprecipitated after incubation of infected cells for 2 or 24 h in the absence and presence of pyrimethamine (PYR), a potent inhibitor of parasite replication.

The Carboxyl Terminus of TgIMC1 Is Proteolytically Processed during Endodyogeny-- In the process of studying the effects of the overexpression of TgIMC1 on the morphology of T. gondii, it was necessary to differentiate TgIMC1 produced under control of other promoters from the endogenous protein. Because we did not know whether insertion of an epitope tag would affect TgIMC1, we generated versions of this protein with either an amino- or a carboxyl-terminal hemagglutinin (HA) tag. As can be seen in Fig. 3, both forms of the protein are expressed well in T. gondii. In the case of the amino-terminal HA-tagged TgIMC1, the protein is found in the subpellicular network of the growing daughter cells and mature parasites, indicating that it behaves analogous to the normal, endogenous TgIMC1. Although TgIMC1 with a carboxyl-terminal HA tag is also targeted efficiently to the network in growing daughter cells (Fig. 3), it is virtually undetectable in the network of mature parasites (Fig. 3). This observation is most easily explained by the proteolytic removal of the carboxyl terminus of TgIMC1 during daughter cell maturation.

View larger version (40K): [in this window] [in a new window]   Fig. 3.   The carboxyl terminus of TgIMC1 is lost during parasite replication. Parasites were transfected with a construct containing full-length TgIMC1 along with either and N-terminal (A, B) or C-terminal (C, D) HA epitope tag. These parasites were then examined using anti-TgIMC1 antibodies (A, C) or anti-HA antibodies (B, D). The labeling pattern of TgIMC1 and the HA tag is identical in parasites containing the N-terminally HA-tagged TgIMC1 (A, B), and both mother and daughter parasites (arrows) are labeled with both antibodies. However, in parasites expressing TgIMC1 with a C-terminal HA tag (C, D), the labeling pattern of TgIMC1 and the HA tag differs with the HA labeling only found in the daughter cells (arrows).

To determine if endogenous TgIMC1 actually undergoes proteolytic processing during parasite maturation and where the cleavage site might be, we performed a pulse-chase experiment followed by immunoprecipitation of TgIMC1. As can be seen in Fig. 4, pulse-labeled TgIMC1 migrates with an apparent molecular mass of 90 kDa, which decreases by about 5 kDa during the chase period. Precursor and mature TgIMC1 are also detectable by immunoblot analysis in intracellular, actively replicating, parasites (see Fig. 7A). In extracellular, non-replicating, parasites only the mature form of TgIMC1 is detectable. Take together with the previous observations, these results indicate that TgIMC1 indeed undergoes proteolytic processing at its carboxyl terminus during maturation of the parasite daughter cells.

View larger version (46K): [in this window] [in a new window]   Fig. 4.   Post-translational proteolytic processing of TgIMC1. Two flasks of Toxoplasma-infected HFF cells were pulse-labeled for 30 min at 37 °C with [35S]methionine and cysteine. One flask (0 h) was placed on ice. The second flask (4 h) was incubated for an additional 4 h after replacement of labeling medium with normal growth medium. Parasites were harvested and lysed in 1% SDS, followed by the immunoprecipitation of TgIMC1 as described under "Experimental Procedures."

It appears that the carboxyl-terminal processing occurs at a very late stage in daughter cell maturation. Even at the stage of endodyogeny when the daughter cells take up most of the mother parasite cytoplasm, the HA tag is still detectable (Fig. 3). Yet we have only very rarely detected the HA tag in mature parasites. This suggests that carboxyl terminal processing of TgIMC1 occurs at the time of or very shortly after emergence of daughter cells from the mother parasite.

Mutational Analysis of TgIMC1 Processing-- Analysis of the predicted amino acid sequence of TgIMC1 had previously revealed that both the amino-terminal and carboxyl-terminal regions of TgIMC1 are relatively rich in cysteine residues (9). The role of these residues and their possible modification by, for example, the addition of lipids has not been determined. To determine whether the carboxyl-terminal cysteine-rich domain (C-CRD) played a role in the proteolytic processing of TgIMC1 during endodyogeny, we generated a mutant TgIMC1 in which amino acids 592-609, comprising the entire C-CRD, was deleted and replaced by the HA tag. As can be seen in Fig. 5, the mutant TgIMC1 was expressed in Toxoplasma and targeted correctly to the inner membrane complex of daughter parasites. But unlike the full-length TgIMC1, the HA tag was not cleaved during maturation of the daughter cells and was present in mature cells. To determine more precisely which residues in the C-CRD are essential for carboxyl-terminal processing, we generated a nested set of carboxyl-terminal deletions of TgIMC1 that were fused to an HA tag. These constructs were transfected into Toxoplasma, and removal of the HA tag during parasite cell division was monitored. As can be seen in Fig. 5, carboxyl-terminal processing is still observed in the absence of residues 595-609 but not in the original deletion mutant in which amino acids 592-609 are removed. This finding implies that one or more of residues 592, 593, and 594, in the sequence Cys-Cys-Val, are essential for carboxyl-terminal processing to occur.

View larger version (48K): [in this window] [in a new window]   Fig. 5.   Identification of the amino acid residues important in carboxyl-terminal proteolytic processing of TgIMC1. A, immunofluorescence analysis of deletion and point mutants using anti-TgIMC1 antibodies shown in the first column, anti-HA antibodies in the second column, and phase contrast microscopy in the third column. B, table summarizes the results of mutational analysis. Mutated residues are underlined.

The role of the individual amino acids in this sequence was determined by the generation of TgIMC1 mutants in which residues 592-596 of the C-CRD were altered individually. As can be seen in Fig. 5, mutation of most residues of this domain did not have a detectable effect on proteolytic processing of TgIMC1 with the exception of the residue 593. Mutation of this cysteine to a threonine (C⁵⁹³T) resulted in a complete loss of carboxyl-terminal processing of TgIMC1, indicating it is essential.

Carboxyl-terminal Processing of TgIMC1 Accompanies Rigidification of the Subpellicular Network-- Carboxyl-terminal proteolytic processing could perform a number of roles in the parasite. However, our observation, that this processing event appears to occur immediately prior to completion of cell division, suggests it plays a role in the final maturation step of Toxoplasma daughter cells. One obvious change that needs to occur in the subpellicular network of replicating parasites upon maturation is the conversion of a plastic structure, which can be expanded and remodeled during growth of the daughter cells and is, therefore, inherently unstable, to a rigid and stable structure that can protect the mature parasite in its extracellular travel from host cell to host cell.

To determine if there is indeed a detectable difference in the properties of the network in mature and immature parasites, we transfected Toxoplasma with a construct containing amino acid residues 1-586 of TgIMC1 fused to YFP. This construct does not undergo carboxyl-terminal proteolytic processing during parasite maturation (Fig. 6A) and can therefore be used to visualize the network in all stages of parasite cell division. We first identified parasites carrying daughter cells microscopically and subsequently extracted these by the addition of deoxycholate (DOC), a detergent that extracts Toxoplasma efficiently but does not affect the network of mature parasites. As can be seen in Fig. 6A, the mother cell network is not affected by the addition of DOC. The network of the daughter cells, on the other hand, is completely extracted. This result demonstrates that the daughter cell network is indeed far more labile than that of the mother cell. This observation was confirmed and quantified by analyzing a large number of isolated intracellular parasites (Fig. 6B). Approximately 12% of these contain TgIMC1-positive daughter cells when fixed prior to detergent extraction. When the parasites are extracted with DOC prior to fixation, however, daughter cells are no longer observed, confirming their sensitivity to detergent extraction and therefore overall stability.

View larger version (78K): [in this window] [in a new window]   Fig. 6.   The subpellicular network of immature and mature parasites differs in stability. A, coverslips of confluent HFF cells were infected overnight with the parasites expressing TgIMC1-(1-586)-YFP, a non-processed form of the TgIMC1 protein. A vacuole containing replicating parasites was identified by fluorescence microscopy (a, b). This same vacuole was re-examined after extraction with 1% DOC (c, d). Disruption of the host cell and parasite-containing vacuoles by DOC is evident as well as the selective extraction of daughter cell network. The network in the mother cell is not affected. B, intracellular parasites were extracted with 1% DOC either before or after methanol fixation. The fraction of parasites that contained TgIMC1-positive daughter cells was determined by immunofluorescence analysis using anti-TgIMC1 antibodies. DOC extraction prior to methanol fixation did not affect TgIMC1 in mature parasites but resulted in a complete loss of TgIMC1-positive daughter cells.

An additional method used to examine any potential differences in the detergent sensitivity of the mature versus the immature forms of TgIMC1 was the extraction of actively replicating intracellular parasites with DOC followed by Western blot analysis of the soluble and insoluble fractions. As can be seen in Fig. 7A, the vast majority of the unprocessed form of TgIMC1 is extracted efficiently by the detergent, whereas processed TgIMC1 is completely detergent-resistant. Similarly, when parasites expressing full-length TgIMC1 carrying a carboxyl-terminal HA tag are extracted with DOC, the vast majority of the total TgIMC1 in the parasites is resistant to extraction (Fig. 7B). The small fraction of TgIMC1, which still carries the HA tag and is therefore part of the immature network in daughter cells, is far more extractable by DOC (Fig. 7B).

View larger version (14K): [in this window] [in a new window]   Fig. 7.   Carboxyl-terminal processing affects the detergent solubility of TgIMC1. A, parasites were extracted with 1% DOC, and the total extract (TOT), and detergent-soluble (SN), and insoluble fractions (P) were examined by immunoblot analysis with anti-TgIMC1 antiserum. B, parasites expressing full-length TgIMC1 with a carboxyl-terminal HA tag were extracted with 1% DOC as above and total (TOT), detergent-soluble (SN), and insoluble (P) fractions were analyzed by immunoblot using antibodies to TgIMC1 and the HA tag.

Taken together, the data in Figs. 6 and 7 demonstrate that the network of immature daughter cells, containing unprocessed TgIMC1, is far more labile than that of mature parasites, which contains processed TgIMC1, and implies that the proteolytic processing at the carboxyl terminus of TgIMC1 may be directly coupled to the conversion of a plastic but labile network to a rigid and stable structure.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

We present evidence that the membrane skeleton of T. gondii, the subpellicular network, is assembled as a labile structure early in the generation of daughter parasites and undergoes a rapid and dramatic increase in stability during the final stage of their maturation. Daughter cells of T. gondii are assembled in the cytoplasm of a mature parasite in a process called endodyogeny. Initially, new inner membrane complexes and cortical microtubule cytoskeletons are assembled in association with two newly generated conoids. These structures grow to envelop a complete set of organelles until eventually the two daughter cells take up most of the volume of the mother parasite. At this stage, the inner membrane complex and cortical microtubules of the mother cell are disassembled or degraded and the remaining plasma membrane collapses onto the daughter cells, which then complete their separation (17-20). As judged by the association of TgIMC1, the subpellicular network of the daughter parasites is assembled at a very early stage in their genesis. Assembly does not appear to involve recycling of components of the mother cell network, because the latter appears intact throughout the development of daughter cells and TgIMC1 of the mother cell seems to be degraded rather than reused. The subpellicular network of immature daughter cells appears to differ substantially from that in mature parasites. Although the network in immature daughter cells is efficiently extracted with detergent, it is completely resistant to extraction in mature daughter cells. This indicates that during maturation of daughter parasites the subpellicular network undergoes a dramatic increase in structural stability.

We propose that the difference in detergent stability reflects a difference in the overall structural stability of the subpellicular network of mature and immature parasites and that this difference is an essential aspect of Toxoplasma cell division. In mature parasites, the subpellicular network is a resilient membrane skeleton that offers mechanical support to the parasite and is composed of stable filaments linked into a network. In immature daughter parasites, however, the network and its filaments would have to be plastic to allow for growth by incorporation of additional subunits and for alterations in its shape. At the last stage in endodyogeny, however, when the inner membrane complex and underlying network of the mother cell are destroyed, the network in the daughter parasites would need to switch rapidly from a loose to a rigid conformation to lend structural support to the newly generated parasites. In this model, Toxoplasma cells would be surrounded by a rigid membrane skeleton at all stages in their development and allow the assembly of daughter cells in a fully protected environment.

The change in detergent solubility during maturation of daughter parasites appears to be accompanied by the proteolytic removal of the carboxyl terminus of TgIMC1. When TgIMC1 containing an amino-terminal HA epitope was expressed in Toxoplasma, the HA epitope was detectable in all developmental stages of the parasite. In contrast, when the HA epitope was attached at the carboxyl terminus of TgIMC1, the HA epitope was detectable in immature daughter cells, up to the stage at which they appear to occupy the majority of the mother cell cytoplasm, but was absent in mature parasites. The simplest interpretation of these observations is that full-length TgIMC1 is incorporated into nascent daughter cells and undergoes carboxyl-terminal proteolytic processing immediately prior to or during breakdown of the mother parasite and emergence of the daughter cells, resulting in loss of the carboxyl-terminal HA epitope. Pulse-chase analysis confirmed that, during maturation of daughter parasites, endogenous TgIMC1 undergoes proteolytic processing resulting a decrease in apparent molecular mass of about 5 kDa. Proteolytic processing of the carboxyl terminus of TgIMC1, as judged by disappearance of the HA epitope, appeared to occur at a stage in daughter cell development close to that where we observed the increase in structural stability of the subpellicular network. To determine whether these events are connected, we analyzed the ability of detergent to solubilize the unprocessed and processed forms of TgIMC1. Detergent effectively solubilized the unprocessed TgIMC1, whereas processed TgIMC1 was entirely detergent-resistant. Similarly, HA-tagged and therefore unprocessed TgIMC1 is easily extracted from parasites. These observations suggest that there is a causal connection between proteolytic processing of TgIMC1 and conversion of the subpellicular network from a plastic into a stable structure.

Deletion and point mutations were generated in TgIMC1 to identify residues required for its carboxyl-terminal proteolytic processing. All mutants tested were targeted normally to the inner membrane complex. The expression of the different mutants did not affect assembly or stability of the subpellicular network, presumably because they were expressed at much lower levels than endogenous TgIMC1 (data not shown). Only a single residue in the C-CRD, Cys⁵⁹³, was found to be essential and alteration of other residues had no discernable effect on processing. These results suggest that either cleavage occurs at Cys⁵⁹³ or that cleavage occurs elsewhere in the carboxyl terminus of TgIMC1 and Cys⁵⁹³ is essential for binding of the processing enzyme. The decrease in molecular weight of TgIMC1 observed during pulse-chase analysis is ~5 kDa, consistent with the removal of about 40 residues. If correct, this would mean that Cys⁵⁹³ lies well downstream of the actual cleavage site and is probably essential for binding of the processing activity. This scenario for TgIMC1 processing is reminiscent of the processing of prelamin A. This nucleoskeleton protein is isoprenylated on a cysteine residue near its carboxyl terminus in the sequence CAAX, followed by proteolytic cleavage behind the modified residue and carboxyl methylation (23, 24). Mature lamin A is generated by a second proteolytic cleavage that removes a carboxyl-terminal peptide that includes the isoprenylated cysteine (25-27). It is tempting to argue that proteolytic processing of TgIMC1 is similar to that of lamin A, in that modification of Cys⁵⁹³ is essential for the subsequent proteolytic processing at a site upstream of that residue. We are in the process of analyzing TgIMC1 processing by both mass spectroscopy and metabolic labeling with lipid precursors to determine both the exact proteolytic cleavage site in TgIMC1 and the covalent modification of the protein and especially Cys⁵⁹³ by lipids.

Two processes are likely to govern the conversion of an instable to a stable membrane skeleton: the actual formation of filaments and their cross-linking into a network. The simplest interpretation of our observations is that the carboxyl terminus of unprocessed TgIMC1 in immature daughter parasites interferes with one or both of these processes and thus maintains a flexible network. Its removal late in endodyogeny would relieve this inhibition and allow the formation of a resilient membrane skeleton. TgIMC1 processing and rigidification of the subpellicular network occur very late in the maturation of daughter parasites and may therefore be coupled to two events that occur at that stage. The inner membrane complex and associated network of the mother parasite are degraded at this time and its plasma membrane collapses onto the inner membrane complex of the daughter cells. TgIMC1 processing and network stabilization could be triggered by either of these processes. Alternatively, TgIMC1 processing and network stabilization could occur first and, upon completion, activate degradation of mother parasite structures. In the first model, one can envision there being a short window during which the mother cell network had been degraded by proteolysis but the daughter cell network had not been made rigid, leaving the daughter parasites in a potentially labile state. For that reason, we favor the latter model at this time, because it provides a mechanism for maintaining the structural integrity of the parasite during all stages of parasite cell division.

Considering the importance of membrane skeletons, such as the subpellicular network, for maintaining the shape and structural integrity of eukaryote cells, the further analysis of their assembly and degradation in Apicomplexan parasites is likely to reveal a number of novel and valuable targets for the development of new strategies to combat these pathogens.

	ACKNOWLEDGEMENTS

We acknowledge Drs. Ke Hu, David Roos, and Gary Ward for many helpful discussions throughout the course of this work.

	FOOTNOTES

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

^¶ To whom correspondence should be addressed: Dept. of Geographic Medicine, University of Alabama, 845 19th St. South, BBRB 206, Birmingham, AL 35294-2170. Tel.: 205-934-1633; Fax: 205-933-5671; E-mail: cbeckers@uab.edu.

Published, JBC Papers in Press, August 12, 2002, DOI 10.1074/jbc.M205056200

	ABBREVIATIONS

The abbreviations used are: HFF, human foreskin fibroblasts; HA, hemagglutinin; YFP, yellow fluorescent protein; DOC, sodium deoxycholate; C-CRD, carboxyl-terminal cysteine-rich domain; PBS, phosphate-buffered saline.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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