Distribution of GPI-anchored proteins in the protozoan parasite Leishmania, based on an improved ultrastructural description using high-pressure frozen cells

F Weise, Y D Stierhof, C Kühn, M Wiese, P Overath

Research output: Contribution to journalArticle

76 Citations (Scopus)

Abstract

The cellular distribution of two glycosyl-phosphatidylinositol (GPI)-anchored proteins and a trans-membrane protein and the compartments involved in their trafficking were investigated in the insect stage of Leishmania mexicana, which belongs to the phylogenetically old protozoan family Trypanosomatidae. Electron microscopy of sections from high-pressure frozen and freeze-substituted cells allowed a detailed description of exo- and endocytic structures located in the vesicle-rich, densely packed anterior part of the spindle-shaped cell. A complex of tubular clusters/translucent vesicles is the prominent structure between the trans-side of the single Golgi apparatus and the flagellar pocket, the only site of endo- and exocytosis. A tubulovesicular compartment lined by one or two distinct microtubules and extending along the length of the cell is proposed to be a post-Golgi and probably late endosomal/lysosomal compartment. Using biotinylation experiments, FACS analysis and quantitative immunoelectron microscopy it was found that, at comparable expression levels, 73-75% of the two GPI-anchored proteins but only 13% of the trans-membrane protein are located on the cell surface. The tubulovesicular compartment contains 46%, the ER 5%, the Golgi complex 1.9% and the tubular cluster/translucent vesicle complex 3.6% of the intracellular fraction of the GPI-anchored protease, GP63. The density of GP63 was found to be 23-fold higher on the plasma/flagellar pocket membrane than on the ER and about tenfold higher than on membranes of the Golgi complex or of endo- or exocytic vesicles. These results indicate that there is a considerable concentration gradient of GPI-anchored proteins between the plasma/flagellar pocket membrane and the ER as well as structures involved in exo- or endocytosis. Possible mechanisms how this concentration gradient is established are discussed.

LanguageEnglish
Pages4587-4603
Number of pages17
JournalJournal of Cell Science
Volume113 Pt 24
Publication statusPublished - Dec 2000

Fingerprint

Protozoan Proteins
Glycosylphosphatidylinositols
Leishmania
Parasites
Golgi Apparatus
Pressure
Exocytosis
Endocytosis
Membranes
Membrane Proteins
Trypanosomatina
Leishmania mexicana
Biotinylation
Immunoelectron Microscopy
Microtubules
Insects
Blood Proteins
Electron Microscopy
Proteins
Peptide Hydrolases

Keywords

  • acid phosphatase
  • antigens, protozoan
  • cell compartmentation
  • cell membranes
  • frozen sections
  • gene expression
  • Glycosylphosphatidylinositols
  • golgi apparatus
  • intracellular membranes
  • Leishmania mexicana
  • metalloendopeptidases
  • microscopy, immunoelectron

Cite this

@article{2fb8a4f8855e479a8a32d06606856c17,
title = "Distribution of GPI-anchored proteins in the protozoan parasite Leishmania, based on an improved ultrastructural description using high-pressure frozen cells",
abstract = "The cellular distribution of two glycosyl-phosphatidylinositol (GPI)-anchored proteins and a trans-membrane protein and the compartments involved in their trafficking were investigated in the insect stage of Leishmania mexicana, which belongs to the phylogenetically old protozoan family Trypanosomatidae. Electron microscopy of sections from high-pressure frozen and freeze-substituted cells allowed a detailed description of exo- and endocytic structures located in the vesicle-rich, densely packed anterior part of the spindle-shaped cell. A complex of tubular clusters/translucent vesicles is the prominent structure between the trans-side of the single Golgi apparatus and the flagellar pocket, the only site of endo- and exocytosis. A tubulovesicular compartment lined by one or two distinct microtubules and extending along the length of the cell is proposed to be a post-Golgi and probably late endosomal/lysosomal compartment. Using biotinylation experiments, FACS analysis and quantitative immunoelectron microscopy it was found that, at comparable expression levels, 73-75{\%} of the two GPI-anchored proteins but only 13{\%} of the trans-membrane protein are located on the cell surface. The tubulovesicular compartment contains 46{\%}, the ER 5{\%}, the Golgi complex 1.9{\%} and the tubular cluster/translucent vesicle complex 3.6{\%} of the intracellular fraction of the GPI-anchored protease, GP63. The density of GP63 was found to be 23-fold higher on the plasma/flagellar pocket membrane than on the ER and about tenfold higher than on membranes of the Golgi complex or of endo- or exocytic vesicles. These results indicate that there is a considerable concentration gradient of GPI-anchored proteins between the plasma/flagellar pocket membrane and the ER as well as structures involved in exo- or endocytosis. Possible mechanisms how this concentration gradient is established are discussed.",
keywords = "acid phosphatase, antigens, protozoan, cell compartmentation, cell membranes, frozen sections, gene expression, Glycosylphosphatidylinositols, golgi apparatus, intracellular membranes, Leishmania mexicana, metalloendopeptidases, microscopy, immunoelectron",
author = "F Weise and Stierhof, {Y D} and C K{\"u}hn and M Wiese and P Overath",
year = "2000",
month = "12",
language = "English",
volume = "113 Pt 24",
pages = "4587--4603",
journal = "Journal of Cell Science",
issn = "0021-9533",

}

Distribution of GPI-anchored proteins in the protozoan parasite Leishmania, based on an improved ultrastructural description using high-pressure frozen cells. / Weise, F; Stierhof, Y D; Kühn, C; Wiese, M; Overath, P.

In: Journal of Cell Science, Vol. 113 Pt 24, 12.2000, p. 4587-4603.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Distribution of GPI-anchored proteins in the protozoan parasite Leishmania, based on an improved ultrastructural description using high-pressure frozen cells

AU - Weise, F

AU - Stierhof, Y D

AU - Kühn, C

AU - Wiese, M

AU - Overath, P

PY - 2000/12

Y1 - 2000/12

N2 - The cellular distribution of two glycosyl-phosphatidylinositol (GPI)-anchored proteins and a trans-membrane protein and the compartments involved in their trafficking were investigated in the insect stage of Leishmania mexicana, which belongs to the phylogenetically old protozoan family Trypanosomatidae. Electron microscopy of sections from high-pressure frozen and freeze-substituted cells allowed a detailed description of exo- and endocytic structures located in the vesicle-rich, densely packed anterior part of the spindle-shaped cell. A complex of tubular clusters/translucent vesicles is the prominent structure between the trans-side of the single Golgi apparatus and the flagellar pocket, the only site of endo- and exocytosis. A tubulovesicular compartment lined by one or two distinct microtubules and extending along the length of the cell is proposed to be a post-Golgi and probably late endosomal/lysosomal compartment. Using biotinylation experiments, FACS analysis and quantitative immunoelectron microscopy it was found that, at comparable expression levels, 73-75% of the two GPI-anchored proteins but only 13% of the trans-membrane protein are located on the cell surface. The tubulovesicular compartment contains 46%, the ER 5%, the Golgi complex 1.9% and the tubular cluster/translucent vesicle complex 3.6% of the intracellular fraction of the GPI-anchored protease, GP63. The density of GP63 was found to be 23-fold higher on the plasma/flagellar pocket membrane than on the ER and about tenfold higher than on membranes of the Golgi complex or of endo- or exocytic vesicles. These results indicate that there is a considerable concentration gradient of GPI-anchored proteins between the plasma/flagellar pocket membrane and the ER as well as structures involved in exo- or endocytosis. Possible mechanisms how this concentration gradient is established are discussed.

AB - The cellular distribution of two glycosyl-phosphatidylinositol (GPI)-anchored proteins and a trans-membrane protein and the compartments involved in their trafficking were investigated in the insect stage of Leishmania mexicana, which belongs to the phylogenetically old protozoan family Trypanosomatidae. Electron microscopy of sections from high-pressure frozen and freeze-substituted cells allowed a detailed description of exo- and endocytic structures located in the vesicle-rich, densely packed anterior part of the spindle-shaped cell. A complex of tubular clusters/translucent vesicles is the prominent structure between the trans-side of the single Golgi apparatus and the flagellar pocket, the only site of endo- and exocytosis. A tubulovesicular compartment lined by one or two distinct microtubules and extending along the length of the cell is proposed to be a post-Golgi and probably late endosomal/lysosomal compartment. Using biotinylation experiments, FACS analysis and quantitative immunoelectron microscopy it was found that, at comparable expression levels, 73-75% of the two GPI-anchored proteins but only 13% of the trans-membrane protein are located on the cell surface. The tubulovesicular compartment contains 46%, the ER 5%, the Golgi complex 1.9% and the tubular cluster/translucent vesicle complex 3.6% of the intracellular fraction of the GPI-anchored protease, GP63. The density of GP63 was found to be 23-fold higher on the plasma/flagellar pocket membrane than on the ER and about tenfold higher than on membranes of the Golgi complex or of endo- or exocytic vesicles. These results indicate that there is a considerable concentration gradient of GPI-anchored proteins between the plasma/flagellar pocket membrane and the ER as well as structures involved in exo- or endocytosis. Possible mechanisms how this concentration gradient is established are discussed.

KW - acid phosphatase

KW - antigens, protozoan

KW - cell compartmentation

KW - cell membranes

KW - frozen sections

KW - gene expression

KW - Glycosylphosphatidylinositols

KW - golgi apparatus

KW - intracellular membranes

KW - Leishmania mexicana

KW - metalloendopeptidases

KW - microscopy, immunoelectron

UR - http://jcs.biologists.org/

UR - http://jcs.biologists.org/content/113/24/4587.abstract

M3 - Article

VL - 113 Pt 24

SP - 4587

EP - 4603

JO - Journal of Cell Science

T2 - Journal of Cell Science

JF - Journal of Cell Science

SN - 0021-9533

ER -