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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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Protein synthesis during BFA treatment assessed by biosynthetic labeling of FtsH1, MIC5 and cytosolic GFP.Fibroblast monolayers infected with T. gondii expressing FtsH1 tagged internally with V5 epitopes and a cytosolic GFP (∼108) were pre-incubated with or without BFA (1 µg/ml) for the indicated times prior to being labeled with 35S-methionine/cysteine for 30 minutes. Samples were immunoprecipitated with anti-V5 mAb, anti-GFP, and anti-MIC5 before being separated on 7.5% (FtsH1) or 8–16% (GFP and MIC5) SDS-PAGE gels and transferred to nitrocellulose. The left panel shows phosphorimaging, the right panel shows the same lanes detected by Western blot. The four major forms of FtsH1 are marked according to their apparent molecular mass on SDS-PAGE: full-length (F-170), N-terminally processed (NP-154), C-terminally processed (CP-140) or dual processed (NPCP-115). In a 30 min labeling, the first two forms predominate [21]. The precursor (p) and mature (m) forms of MIC5 [75] are marked.
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pone-0112096-g004: Protein synthesis during BFA treatment assessed by biosynthetic labeling of FtsH1, MIC5 and cytosolic GFP.Fibroblast monolayers infected with T. gondii expressing FtsH1 tagged internally with V5 epitopes and a cytosolic GFP (∼108) were pre-incubated with or without BFA (1 µg/ml) for the indicated times prior to being labeled with 35S-methionine/cysteine for 30 minutes. Samples were immunoprecipitated with anti-V5 mAb, anti-GFP, and anti-MIC5 before being separated on 7.5% (FtsH1) or 8–16% (GFP and MIC5) SDS-PAGE gels and transferred to nitrocellulose. The left panel shows phosphorimaging, the right panel shows the same lanes detected by Western blot. The four major forms of FtsH1 are marked according to their apparent molecular mass on SDS-PAGE: full-length (F-170), N-terminally processed (NP-154), C-terminally processed (CP-140) or dual processed (NPCP-115). In a 30 min labeling, the first two forms predominate [21]. The precursor (p) and mature (m) forms of MIC5 [75] are marked.

Mentions: ApV proteins could transit very rapidly through the Golgi body, thus escaping steady state detection. We therefore examined in detail the effect of BFA treatment on the presence of Vap. If Vap represented ER to Golgi or Golgi to apicoplast intermediates, we would expect that a block of Golgi function would inhibit their formation. Although the Golgi marker NST1 relocalized to the ER within 15 min of the application of BFA (not shown), the drug might not affect the trafficking of previously formed Golgi to apicoplast intermediates. Thus, we aimed to incubate the parasites in drug as long as possible to allow pre-existing Vap to arrive at their destination while still allowing protein synthesis to generate new Vap cargo. Protein synthesis, assessed by 35S-methionine labeling of three proteins (FtsH1, the microneme protein MIC5, and cytosolic GFP), continued robustly for 1.5 hour after application of BFA, being very similar to the untreated control (Fig. 4). Subsequently, protein synthesis dropped precipitously in the BFA-treated parasites. Therefore we chose a 1.5 hour treatment with BFA for our IFA studies.


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)

Protein synthesis during BFA treatment assessed by biosynthetic labeling of FtsH1, MIC5 and cytosolic GFP.Fibroblast monolayers infected with T. gondii expressing FtsH1 tagged internally with V5 epitopes and a cytosolic GFP (∼108) were pre-incubated with or without BFA (1 µg/ml) for the indicated times prior to being labeled with 35S-methionine/cysteine for 30 minutes. Samples were immunoprecipitated with anti-V5 mAb, anti-GFP, and anti-MIC5 before being separated on 7.5% (FtsH1) or 8–16% (GFP and MIC5) SDS-PAGE gels and transferred to nitrocellulose. The left panel shows phosphorimaging, the right panel shows the same lanes detected by Western blot. The four major forms of FtsH1 are marked according to their apparent molecular mass on SDS-PAGE: full-length (F-170), N-terminally processed (NP-154), C-terminally processed (CP-140) or dual processed (NPCP-115). In a 30 min labeling, the first two forms predominate [21]. The precursor (p) and mature (m) forms of MIC5 [75] are marked.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4219833&req=5

pone-0112096-g004: Protein synthesis during BFA treatment assessed by biosynthetic labeling of FtsH1, MIC5 and cytosolic GFP.Fibroblast monolayers infected with T. gondii expressing FtsH1 tagged internally with V5 epitopes and a cytosolic GFP (∼108) were pre-incubated with or without BFA (1 µg/ml) for the indicated times prior to being labeled with 35S-methionine/cysteine for 30 minutes. Samples were immunoprecipitated with anti-V5 mAb, anti-GFP, and anti-MIC5 before being separated on 7.5% (FtsH1) or 8–16% (GFP and MIC5) SDS-PAGE gels and transferred to nitrocellulose. The left panel shows phosphorimaging, the right panel shows the same lanes detected by Western blot. The four major forms of FtsH1 are marked according to their apparent molecular mass on SDS-PAGE: full-length (F-170), N-terminally processed (NP-154), C-terminally processed (CP-140) or dual processed (NPCP-115). In a 30 min labeling, the first two forms predominate [21]. The precursor (p) and mature (m) forms of MIC5 [75] are marked.
Mentions: ApV proteins could transit very rapidly through the Golgi body, thus escaping steady state detection. We therefore examined in detail the effect of BFA treatment on the presence of Vap. If Vap represented ER to Golgi or Golgi to apicoplast intermediates, we would expect that a block of Golgi function would inhibit their formation. Although the Golgi marker NST1 relocalized to the ER within 15 min of the application of BFA (not shown), the drug might not affect the trafficking of previously formed Golgi to apicoplast intermediates. Thus, we aimed to incubate the parasites in drug as long as possible to allow pre-existing Vap to arrive at their destination while still allowing protein synthesis to generate new Vap cargo. Protein synthesis, assessed by 35S-methionine labeling of three proteins (FtsH1, the microneme protein MIC5, and cytosolic GFP), continued robustly for 1.5 hour after application of BFA, being very similar to the untreated control (Fig. 4). Subsequently, protein synthesis dropped precipitously in the BFA-treated parasites. Therefore we chose a 1.5 hour treatment with BFA for our IFA studies.

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.

Show MeSH
Related in: MedlinePlus