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

The cellular distribution of two glycosylphosphatidylinositol (GPI)-anchore d proteins and a trans-membrane protein and the compartments involved in th eir trafficking were investigated in the insect stage of Leishmania mexican a, which belongs to the phylogenetically old protozoan family Trypanosomati dae. Electron microscopy of sections from high-pressure frozen and freeze-s ubstituted cells allowed a detailed description of exo- and endocytic struc tures located in the vesicle-rich, densely packed anterior part of the spin dle-shaped cell. A complex of tubular clusters/translucent vesicles is the prominent structure between the trans-side of the single Golgi apparatus an d the flagellar pocket, the only site of endo- and exocytosis. A tubulovesi cular compartment lined by one or two distinct microtubules and extending a long the length of the cell is proposed to be a post-Golgi and probably lat e endosomal/lysosomal compartment. Using biotinylation experiments, FAGS an alysis and quantitative immunoelectron microscopy it was found that, at com parable 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 tub ulovesicular compartment contains 46%, the ER 5%, the Golgi complex 1.9% an d 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 E R and about tenfold higher than on membranes of the Golgi complex or of end o- or exocytic vesicles. These results indicate that there is a considerabl e concentration gradient of GPI-anchored proteins between the plasma/flagel lar pocket membrane and the ER as well as structures involved in exo- or en docytosis. Possible mechanisms how this concentration gradient is establish ed are discussed.