Isolation and characterization of a highly purified flagellar membrane fraction from trypanosomatids

Cunha-e-Silva, Narcisa L.; A, Hassón-Voloch; Souza, Wanderley de

doi:10.1016/0166-6851(89)90109-6

Cited by 20 publications

(8 citation statements)

References 20 publications

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“…Cardiolipin and PG are confined to mitochondria in eukaryotic cells. Accordingly, using subcellular fractionation, immunohistochemistry and immunofluorescence microscopy, Cls and Pgps have been localized to (inner) mitochondrial membranes in S. cerevisiae (da Cunha e Silva et al ., ; Dzugasova et al ., ) and mammalian cells (Furlong, ; Schlame and Haldar, ; Denny et al ., ; Nes et al ., ). Similarly, in a recent study functionally active, epitope‐tagged TbCls was localized to the mitochondrion in T. brucei procyclic forms (Serricchio and Bütikofer, ).…”

Section: Discussionmentioning

confidence: 98%

Phosphatidylglycerophosphate synthase associates with a mitochondrial inner membrane complex and is essential for growth of Trypanosoma brucei

Serricchio

Bütikofer

2012

Molecular Microbiology

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SummaryMaintenance of the lipid composition is important for proper function and homeostasis of the mitochondrion. In Trypanosoma brucei, the enzymes involved in the biosynthesis of the mitochondrial phospholipid, phosphatidylglycerol (PG), have not been studied experimentally. We now report the characterization of T. brucei phosphatidylglycerophosphate synthase (TbPgps), the rate-limiting enzyme in PG formation, which was identified based on its homology to other eukaryotic Pgps. Lipid quantification and metabolic labelling experiments show that TbPgps gene knockdown results in loss of PG and a reduction of another mitochondria-specific phospholipid, cardiolipin. Using immunohistochemistry and immunoblotting of digitonin-isolated mitochondria, we show that TbPgps localizes to the mitochondrion. Moreover, reduced TbPgps expression in T. brucei procyclic forms leads to alterations in mitochondrial morphology, reduction in the amounts of respiratory complexes III and IV and, ultimately, parasite death. Using native polyacrylamide gel electrophoresis we demonstrate for the first time in a eukaryotic organism that TbPgps is a component of a 720 kDa protein complex, co-migrating with T. brucei cardiolipin synthase and cytochrome c1, a protein of respiratory complex III.

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Section: Discussionmentioning

confidence: 98%

Phosphatidylglycerophosphate synthase associates with a mitochondrial inner membrane complex and is essential for growth of Trypanosoma brucei

Serricchio

Bütikofer

2012

Molecular Microbiology

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“…The observation that the bi-lobed structure was tightly associated with the flagellum, even after detergent extraction and high-salt wash, suggests that it is a core component of the flagellar complex, which also includes the basal bodies that seed flagellum growth and the FPC that is required for flagellar pocket biogenesis. Using a modified cell fractionation protocol that was first developed by da Cunha e Silva et al [19], flagella detached from cell bodies and therefore free of associated basal bodies or bi-lobes were purified from T. brucei . The flagellar complex and the purified flagella samples were then analyzed by iTRAQ, which allowed the relative abundance of each protein to be calculated leading to the identification of proteins present only in the flagellar complex but not in the detached flagella.…”

Section: Discussionmentioning

confidence: 99%

“…Flagellum free of basal bodies and bi-lobes was purified using a procedure described previously [19] with the following modification. Briefly, 3×10 9 procyclic cells were harvested, washed and resuspended in homogenization buffer containing 0.32 M sucrose, 25 mM Tris HCl, pH 7.4, 5 mM MgCl 2 , 0.1 mM CaCl 2 , 0.2 mM EDTA, 12 mM beta-mercaptoethanol and 1% bovine serum albumin.…”

Section: Methodsmentioning

confidence: 99%

A Comparative Proteomic Analysis Reveals a New Bi-Lobe Protein Required for Bi-Lobe Duplication and Cell Division in Trypanosoma brucei

et al. 2010

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A Golgi-associated bi-lobed structure was previously found to be important for Golgi duplication and cell division in Trypanosoma brucei. To further understand its functions, comparative proteomics was performed on extracted flagellar complexes (including the flagellum and flagellum-associated structures such as the basal bodies and the bi-lobe) and purified flagella to identify new bi-lobe proteins. A leucine-rich repeats containing protein, TbLRRP1, was characterized as a new bi-lobe component. The anterior part of the TbLRRP1-labeled bi-lobe is adjacent to the single Golgi apparatus, and the posterior side is tightly associated with the flagellar pocket collar marked by TbBILBO1. Inducible depletion of TbLRRP1 by RNA interference inhibited duplication of the bi-lobe as well as the adjacent Golgi apparatus and flagellar pocket collar. Formation of a new flagellum attachment zone and subsequent cell division were also inhibited, suggesting a central role of bi-lobe in Golgi, flagellar pocket collar and flagellum attachment zone biogenesis.

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“…Flagella were purified essentially as previously described (da Cunha e Silva et al, 1989). Briefly, 10 10 trypanosomes were washed and resuspended in buffer A (25 mM tricine, 0.2 mM EDTA, 5 mM MgCl 2 , 12 mM β-mercaptoethanol, pH 7.0).…”

Section: Purification Of Flagellamentioning

confidence: 99%

Flagellar membrane localization via association with lipid rafts

Fridberg

Toriello

Olson

et al. 2009

Journal of Cell Science

121

131

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the lipid rafts are disrupted. Detergent-extracted cells have discrete membrane patches localized on the surface of the flagellar axoneme, suggestive of intraflagellar transport particles. Together, these results provide biophysical and biochemical evidence to indicate that lipid rafts are enriched in the trypanosome flagellar membrane, providing a unique mechanism for flagellar protein localization and illustrating a novel means by which specialized cellular functions may be partitioned to discrete membrane domains.

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Isolation and characterization of a highly purified flagellar membrane fraction from trypanosomatids

Cited by 20 publications

References 20 publications

Phosphatidylglycerophosphate synthase associates with a mitochondrial inner membrane complex and is essential for growth of Trypanosoma brucei

Phosphatidylglycerophosphate synthase associates with a mitochondrial inner membrane complex and is essential for growth of Trypanosoma brucei

A Comparative Proteomic Analysis Reveals a New Bi-Lobe Protein Required for Bi-Lobe Duplication and Cell Division in Trypanosoma brucei

Flagellar membrane localization via association with lipid rafts

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