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Vesicles bearing Toxoplasma apicoplast membrane proteins persist following loss of the relict plastid or Golgi body disruption.

Bouchut A, Geiger JA, DeRocher AE, Parsons M - PLoS ONE (2014)

Bottom Line: The immunofluorescence patterns showed little change.These findings were confirmed using stable transfectants, which expressed the toxic dominant-negative sar1 following Cre-loxP mediated promoter juxtaposition.These data raise the possibility that the apicoplast proteome is generated by two novel ER to plastid trafficking pathways, plus the small set of proteins encoded by the apicoplast genome.

View Article: PubMed Central - PubMed

Affiliation: Seattle Biomedical Research Institute, Seattle, WA, United States of America.

ABSTRACT
Toxoplasma gondii and malaria parasites contain a unique and essential relict plastid called the apicoplast. Most apicoplast proteins are encoded in the nucleus and are transported to the organelle via the endoplasmic reticulum (ER). Three trafficking routes have been proposed for apicoplast membrane proteins: (i) vesicular trafficking from the ER to the Golgi and then to the apicoplast, (ii) contiguity between the ER membrane and the apicoplast allowing direct flow of proteins, and (iii) vesicular transport directly from the ER to the apicoplast. Previously, we identified a set of membrane proteins of the T. gondii apicoplast which were also detected in large vesicles near the organelle. Data presented here show that the large vesicles bearing apicoplast membrane proteins are not the major carriers of luminal proteins. The vesicles continue to appear in parasites which have lost their plastid due to mis-segregation, indicating that the vesicles are not derived from the apicoplast. To test for a role of the Golgi body in vesicle formation, parasites were treated with brefeldin A or transiently transfected with a dominant-negative mutant of Sar1, a GTPase required for ER to Golgi trafficking. The immunofluorescence patterns showed little change. These findings were confirmed using stable transfectants, which expressed the toxic dominant-negative sar1 following Cre-loxP mediated promoter juxtaposition. Our data support the hypothesis that the large vesicles do not mediate the trafficking of luminal proteins to the apicoplast. The results further show that the large vesicles bearing apicoplast membrane proteins continue to be observed in the absence of Golgi and plastid function. These data raise the possibility that the apicoplast proteome is generated by two novel ER to plastid trafficking pathways, plus the small set of proteins encoded by the apicoplast genome.

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Vap persist in parasites with plastid loss.T. gondii expressing the indicated tagged apicoplast proteins were transiently transfected with a plasmid encoding S+TYFP-ROP1 (chimera, endogenous fluorescence) to induce plastid mis-segregation. After 40 hours to allow for apicoplast loss through several cell divisions, the samples were subjected to IFA. Vacuoles with one or more parasites expressing the chimeric protein were analyzed. Individual cells and vacuoles are outlined with solid lines and dashed lines respectively. The markers are indicated above each panel. DIC, differential interference contrast, H indicates host cell nucleus. A) Loss of luminal marker in parasites expressing the “poison” chimera. S+TRed-V5 was detected with both anti-V5 mAb (followed by secondary antibody coupled to Dylight 649; panels labeled S+TRed-V5), and through intrinsic fluorescence (panels here and in B, C labeled S+TRed). The lower panels show enhanced scaling of S+TRed-V5 detected with anti-V5 to highlight faint ER-like staining. Bar = 5 µM. B) Continued formation of Vap bearing FtsH1. FtsH1/S+TRed parasites were transiently transfected with the chimeric construct (detected by endogenous fluorescence) and FtsH1 was detected with anti-V5 mAb (followed by secondary antibody coupled to DyLight 649). Background of the S+TRed images in panels B and C were adjusted to correct for crossover fluorescence from the DyLight 649 fluorophore. Arrows indicate Vap-like staining in cells lacking an apicoplast. Vacuoles bearing transfected parasites (upper left) and untransfected parasites (lower right) are shown. Bar = 5 µM. C) Continued formation of Vap bearing ATrx1. ATrx1/S+TRed expressing cells were transiently transfected with S+TROP1-YFP, which was detected by endogenous fluorescence. ATrx1 was detected with anti-HA mAb coupled to DyLight 649. Arrows indicate Vap-like staining in cells lacking an apicoplast. Vacuoles bearing transfected parasites (upper left) and untransfected parasites (lower right) are shown. Parasites in the lower vacuole are in stage 1 of the organelle division cycle and therefore have few Vap. Bar = 5 µM. D) Quantitation of Vap in apicoplast-deficient parasites. ATrx1-4HA/S+TRed and FtsH1-V5233-HA/S+TRed expressing cell lines were transiently transfected with the chimeric S+TYFP-ROP1 construct and vacuoles were scored for the presence or absence of YFP in at least one parasite (indicating expression of the chimeric protein in the original invading parasite). Individual parasites within each vacuole were then scored for the presence or absence of the luminal protein S+TRed (detected by endogenous fluorescence) and the apicoplast membrane protein (detected by anti-HA or anti-V5 mAbs followed by anti-mouse IgG coupled to DyLight 649). The bar graph plots the percentage of cells bearing each marker protein in vacuoles derived from transfected (chimera+) and untransfected (chimera−) parasites. In the ATrx1 sample, 96 chimera+ and 128 chimera− cells were counted; in the FtsH1 sample, 27 chimera+ and 48 chimera− cells were counted. These results are representative of three independent experiments.
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pone-0112096-g002: Vap persist in parasites with plastid loss.T. gondii expressing the indicated tagged apicoplast proteins were transiently transfected with a plasmid encoding S+TYFP-ROP1 (chimera, endogenous fluorescence) to induce plastid mis-segregation. After 40 hours to allow for apicoplast loss through several cell divisions, the samples were subjected to IFA. Vacuoles with one or more parasites expressing the chimeric protein were analyzed. Individual cells and vacuoles are outlined with solid lines and dashed lines respectively. The markers are indicated above each panel. DIC, differential interference contrast, H indicates host cell nucleus. A) Loss of luminal marker in parasites expressing the “poison” chimera. S+TRed-V5 was detected with both anti-V5 mAb (followed by secondary antibody coupled to Dylight 649; panels labeled S+TRed-V5), and through intrinsic fluorescence (panels here and in B, C labeled S+TRed). The lower panels show enhanced scaling of S+TRed-V5 detected with anti-V5 to highlight faint ER-like staining. Bar = 5 µM. B) Continued formation of Vap bearing FtsH1. FtsH1/S+TRed parasites were transiently transfected with the chimeric construct (detected by endogenous fluorescence) and FtsH1 was detected with anti-V5 mAb (followed by secondary antibody coupled to DyLight 649). Background of the S+TRed images in panels B and C were adjusted to correct for crossover fluorescence from the DyLight 649 fluorophore. Arrows indicate Vap-like staining in cells lacking an apicoplast. Vacuoles bearing transfected parasites (upper left) and untransfected parasites (lower right) are shown. Bar = 5 µM. C) Continued formation of Vap bearing ATrx1. ATrx1/S+TRed expressing cells were transiently transfected with S+TROP1-YFP, which was detected by endogenous fluorescence. ATrx1 was detected with anti-HA mAb coupled to DyLight 649. Arrows indicate Vap-like staining in cells lacking an apicoplast. Vacuoles bearing transfected parasites (upper left) and untransfected parasites (lower right) are shown. Parasites in the lower vacuole are in stage 1 of the organelle division cycle and therefore have few Vap. Bar = 5 µM. D) Quantitation of Vap in apicoplast-deficient parasites. ATrx1-4HA/S+TRed and FtsH1-V5233-HA/S+TRed expressing cell lines were transiently transfected with the chimeric S+TYFP-ROP1 construct and vacuoles were scored for the presence or absence of YFP in at least one parasite (indicating expression of the chimeric protein in the original invading parasite). Individual parasites within each vacuole were then scored for the presence or absence of the luminal protein S+TRed (detected by endogenous fluorescence) and the apicoplast membrane protein (detected by anti-HA or anti-V5 mAbs followed by anti-mouse IgG coupled to DyLight 649). The bar graph plots the percentage of cells bearing each marker protein in vacuoles derived from transfected (chimera+) and untransfected (chimera−) parasites. In the ATrx1 sample, 96 chimera+ and 128 chimera− cells were counted; in the FtsH1 sample, 27 chimera+ and 48 chimera− cells were counted. These results are representative of three independent experiments.

Mentions: To further compare ApV proteins to luminal proteins, we examined their localization in T. gondii lacking an apicoplast. These parasites were generated by using a “poison” construct described by He et al. [30], which encodes a chimeric protein composed of an apicoplast targeting sequence fused to YFP followed by the mature domain of the rhoptry protein Rop1 (S+TYFP-ROP1). In previous studies, it was shown that the chimeric protein targets to the apicoplast and disrupts plastid segregation, often resulting in parasitophorous vacuoles containing one cell with a large plastid and several cells apparently lacking apicoplast luminal proteins as well as the apicoplast genome [30]. The plasmid was transiently transfected into cells expressing a red fluorescent luminal protein marker (S+TRed or S+TRed-V5) along with tagged ApV proteins ATrx1 or FtsH1. Our analysis focused on those vacuoles with strong expression of the chimeric protein in only one parasite. There was a marked difference in the fate of the luminal and ApV proteins in cells lacking an apicoplast (Fig. 2). As expected, the apicoplast luminal marker partitioned with the chimeric protein and these were either localized together typically at the apicoplast (but occasionally at the residual body), or not detected at all by intrinsic fluorescence. Using anti-V5 antibody to visualize S+TRed-V5 prior to chromophore maturation additionally revealed the protein in a faint ER-like pattern in some cells (Fig. 2A, “enhanced”), suggesting continued S+TRed-V5 production. This pattern appeared to be somewhat more frequent in parasites that lacked an apicoplast, although the difference from control was not statistically significant. ATrx1 and FtsH1 on the other hand accumulated in structures apical to the nucleus (examples indicated by arrows), similar to the Vap seen in the cells with an apicoplast (Fig. 2B, C). Quantitative analysis of progeny of parasites expressing the chimeric construct showed that only about 20% stained for the luminal marker (Fig. 2D). In contrast almost all parasites had Vap as revealed by ATrx1 or FtsH1 markers, whether or not the vacuoles were positive for the chimeric protein. These findings corroborate a previous study in which the apicoplast was rapidly eliminated but Vap retained following expression of a PI3P-binding protein [27]. Taken together, the above data supports the possibility of two trafficking pathways: one for luminal proteins and one for ApV proteins. Furthermore, the similar abundance of Vap bearing ATrx1 and FtsH1 in cells with and without an apicoplast indicates that Vap do not arise from apicoplast.


Vesicles bearing Toxoplasma apicoplast membrane proteins persist following loss of the relict plastid or Golgi body disruption.

Bouchut A, Geiger JA, DeRocher AE, Parsons M - PLoS ONE (2014)

Vap persist in parasites with plastid loss.T. gondii expressing the indicated tagged apicoplast proteins were transiently transfected with a plasmid encoding S+TYFP-ROP1 (chimera, endogenous fluorescence) to induce plastid mis-segregation. After 40 hours to allow for apicoplast loss through several cell divisions, the samples were subjected to IFA. Vacuoles with one or more parasites expressing the chimeric protein were analyzed. Individual cells and vacuoles are outlined with solid lines and dashed lines respectively. The markers are indicated above each panel. DIC, differential interference contrast, H indicates host cell nucleus. A) Loss of luminal marker in parasites expressing the “poison” chimera. S+TRed-V5 was detected with both anti-V5 mAb (followed by secondary antibody coupled to Dylight 649; panels labeled S+TRed-V5), and through intrinsic fluorescence (panels here and in B, C labeled S+TRed). The lower panels show enhanced scaling of S+TRed-V5 detected with anti-V5 to highlight faint ER-like staining. Bar = 5 µM. B) Continued formation of Vap bearing FtsH1. FtsH1/S+TRed parasites were transiently transfected with the chimeric construct (detected by endogenous fluorescence) and FtsH1 was detected with anti-V5 mAb (followed by secondary antibody coupled to DyLight 649). Background of the S+TRed images in panels B and C were adjusted to correct for crossover fluorescence from the DyLight 649 fluorophore. Arrows indicate Vap-like staining in cells lacking an apicoplast. Vacuoles bearing transfected parasites (upper left) and untransfected parasites (lower right) are shown. Bar = 5 µM. C) Continued formation of Vap bearing ATrx1. ATrx1/S+TRed expressing cells were transiently transfected with S+TROP1-YFP, which was detected by endogenous fluorescence. ATrx1 was detected with anti-HA mAb coupled to DyLight 649. Arrows indicate Vap-like staining in cells lacking an apicoplast. Vacuoles bearing transfected parasites (upper left) and untransfected parasites (lower right) are shown. Parasites in the lower vacuole are in stage 1 of the organelle division cycle and therefore have few Vap. Bar = 5 µM. D) Quantitation of Vap in apicoplast-deficient parasites. ATrx1-4HA/S+TRed and FtsH1-V5233-HA/S+TRed expressing cell lines were transiently transfected with the chimeric S+TYFP-ROP1 construct and vacuoles were scored for the presence or absence of YFP in at least one parasite (indicating expression of the chimeric protein in the original invading parasite). Individual parasites within each vacuole were then scored for the presence or absence of the luminal protein S+TRed (detected by endogenous fluorescence) and the apicoplast membrane protein (detected by anti-HA or anti-V5 mAbs followed by anti-mouse IgG coupled to DyLight 649). The bar graph plots the percentage of cells bearing each marker protein in vacuoles derived from transfected (chimera+) and untransfected (chimera−) parasites. In the ATrx1 sample, 96 chimera+ and 128 chimera− cells were counted; in the FtsH1 sample, 27 chimera+ and 48 chimera− cells were counted. These results are representative of three independent experiments.
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pone-0112096-g002: Vap persist in parasites with plastid loss.T. gondii expressing the indicated tagged apicoplast proteins were transiently transfected with a plasmid encoding S+TYFP-ROP1 (chimera, endogenous fluorescence) to induce plastid mis-segregation. After 40 hours to allow for apicoplast loss through several cell divisions, the samples were subjected to IFA. Vacuoles with one or more parasites expressing the chimeric protein were analyzed. Individual cells and vacuoles are outlined with solid lines and dashed lines respectively. The markers are indicated above each panel. DIC, differential interference contrast, H indicates host cell nucleus. A) Loss of luminal marker in parasites expressing the “poison” chimera. S+TRed-V5 was detected with both anti-V5 mAb (followed by secondary antibody coupled to Dylight 649; panels labeled S+TRed-V5), and through intrinsic fluorescence (panels here and in B, C labeled S+TRed). The lower panels show enhanced scaling of S+TRed-V5 detected with anti-V5 to highlight faint ER-like staining. Bar = 5 µM. B) Continued formation of Vap bearing FtsH1. FtsH1/S+TRed parasites were transiently transfected with the chimeric construct (detected by endogenous fluorescence) and FtsH1 was detected with anti-V5 mAb (followed by secondary antibody coupled to DyLight 649). Background of the S+TRed images in panels B and C were adjusted to correct for crossover fluorescence from the DyLight 649 fluorophore. Arrows indicate Vap-like staining in cells lacking an apicoplast. Vacuoles bearing transfected parasites (upper left) and untransfected parasites (lower right) are shown. Bar = 5 µM. C) Continued formation of Vap bearing ATrx1. ATrx1/S+TRed expressing cells were transiently transfected with S+TROP1-YFP, which was detected by endogenous fluorescence. ATrx1 was detected with anti-HA mAb coupled to DyLight 649. Arrows indicate Vap-like staining in cells lacking an apicoplast. Vacuoles bearing transfected parasites (upper left) and untransfected parasites (lower right) are shown. Parasites in the lower vacuole are in stage 1 of the organelle division cycle and therefore have few Vap. Bar = 5 µM. D) Quantitation of Vap in apicoplast-deficient parasites. ATrx1-4HA/S+TRed and FtsH1-V5233-HA/S+TRed expressing cell lines were transiently transfected with the chimeric S+TYFP-ROP1 construct and vacuoles were scored for the presence or absence of YFP in at least one parasite (indicating expression of the chimeric protein in the original invading parasite). Individual parasites within each vacuole were then scored for the presence or absence of the luminal protein S+TRed (detected by endogenous fluorescence) and the apicoplast membrane protein (detected by anti-HA or anti-V5 mAbs followed by anti-mouse IgG coupled to DyLight 649). The bar graph plots the percentage of cells bearing each marker protein in vacuoles derived from transfected (chimera+) and untransfected (chimera−) parasites. In the ATrx1 sample, 96 chimera+ and 128 chimera− cells were counted; in the FtsH1 sample, 27 chimera+ and 48 chimera− cells were counted. These results are representative of three independent experiments.
Mentions: To further compare ApV proteins to luminal proteins, we examined their localization in T. gondii lacking an apicoplast. These parasites were generated by using a “poison” construct described by He et al. [30], which encodes a chimeric protein composed of an apicoplast targeting sequence fused to YFP followed by the mature domain of the rhoptry protein Rop1 (S+TYFP-ROP1). In previous studies, it was shown that the chimeric protein targets to the apicoplast and disrupts plastid segregation, often resulting in parasitophorous vacuoles containing one cell with a large plastid and several cells apparently lacking apicoplast luminal proteins as well as the apicoplast genome [30]. The plasmid was transiently transfected into cells expressing a red fluorescent luminal protein marker (S+TRed or S+TRed-V5) along with tagged ApV proteins ATrx1 or FtsH1. Our analysis focused on those vacuoles with strong expression of the chimeric protein in only one parasite. There was a marked difference in the fate of the luminal and ApV proteins in cells lacking an apicoplast (Fig. 2). As expected, the apicoplast luminal marker partitioned with the chimeric protein and these were either localized together typically at the apicoplast (but occasionally at the residual body), or not detected at all by intrinsic fluorescence. Using anti-V5 antibody to visualize S+TRed-V5 prior to chromophore maturation additionally revealed the protein in a faint ER-like pattern in some cells (Fig. 2A, “enhanced”), suggesting continued S+TRed-V5 production. This pattern appeared to be somewhat more frequent in parasites that lacked an apicoplast, although the difference from control was not statistically significant. ATrx1 and FtsH1 on the other hand accumulated in structures apical to the nucleus (examples indicated by arrows), similar to the Vap seen in the cells with an apicoplast (Fig. 2B, C). Quantitative analysis of progeny of parasites expressing the chimeric construct showed that only about 20% stained for the luminal marker (Fig. 2D). In contrast almost all parasites had Vap as revealed by ATrx1 or FtsH1 markers, whether or not the vacuoles were positive for the chimeric protein. These findings corroborate a previous study in which the apicoplast was rapidly eliminated but Vap retained following expression of a PI3P-binding protein [27]. Taken together, the above data supports the possibility of two trafficking pathways: one for luminal proteins and one for ApV proteins. Furthermore, the similar abundance of Vap bearing ATrx1 and FtsH1 in cells with and without an apicoplast indicates that Vap do not arise from apicoplast.

Bottom Line: The immunofluorescence patterns showed little change.These findings were confirmed using stable transfectants, which expressed the toxic dominant-negative sar1 following Cre-loxP mediated promoter juxtaposition.These data raise the possibility that the apicoplast proteome is generated by two novel ER to plastid trafficking pathways, plus the small set of proteins encoded by the apicoplast genome.

View Article: PubMed Central - PubMed

Affiliation: Seattle Biomedical Research Institute, Seattle, WA, United States of America.

ABSTRACT
Toxoplasma gondii and malaria parasites contain a unique and essential relict plastid called the apicoplast. Most apicoplast proteins are encoded in the nucleus and are transported to the organelle via the endoplasmic reticulum (ER). Three trafficking routes have been proposed for apicoplast membrane proteins: (i) vesicular trafficking from the ER to the Golgi and then to the apicoplast, (ii) contiguity between the ER membrane and the apicoplast allowing direct flow of proteins, and (iii) vesicular transport directly from the ER to the apicoplast. Previously, we identified a set of membrane proteins of the T. gondii apicoplast which were also detected in large vesicles near the organelle. Data presented here show that the large vesicles bearing apicoplast membrane proteins are not the major carriers of luminal proteins. The vesicles continue to appear in parasites which have lost their plastid due to mis-segregation, indicating that the vesicles are not derived from the apicoplast. To test for a role of the Golgi body in vesicle formation, parasites were treated with brefeldin A or transiently transfected with a dominant-negative mutant of Sar1, a GTPase required for ER to Golgi trafficking. The immunofluorescence patterns showed little change. These findings were confirmed using stable transfectants, which expressed the toxic dominant-negative sar1 following Cre-loxP mediated promoter juxtaposition. Our data support the hypothesis that the large vesicles do not mediate the trafficking of luminal proteins to the apicoplast. The results further show that the large vesicles bearing apicoplast membrane proteins continue to be observed in the absence of Golgi and plastid function. These data raise the possibility that the apicoplast proteome is generated by two novel ER to plastid trafficking pathways, plus the small set of proteins encoded by the apicoplast genome.

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Related in: MedlinePlus