The Glu-modification of alpha-tubulin in the feeding apparatus of the primitive flagellate Entosiphon sulcatum is only apparent after detergent treatment.
Using specific monoclonal antibodies, we investigated the distribution of post-translational modified Tyr- and Glu-tubulins during interphase of the primitive flagellate Entosiphon sulcatum. Immunofluorescence studies of simultaneously permeabilized and fixed cells revealed that microtubular structures comprising Ca(2+)-labile subpellicular and flagellar MTs and Ca(2+)-stable MTs in the siphon complex (feeding organelle) reacted surprisingly unorthodox with antibodies against Tyr- and Glu-tubulin: Unexpectedly, the siphon complex consisting of Ca(2+)-stable MTs appeared exclusively Tyr-positive, whereas the Ca(2+)-labile subpellicular and flagellar MTs reacted with the Glu- as well as with the Tyr-antibody. That the siphon MTs were indeed Ca(2+)-stable and all other MTs had become solubilized, was verified by EM-observation. This surprising result contrasting considerably with the permanent nature of the siphon complex, was reconsidered after preceding lysis and extraction procedures. Depending on the type of detergent used and on extraction times applied, the MTs of the siphon complex now always showed also Glu-positivity, indicating the presence of detyrosinated alpha-tubulin as a biochemical marker of stabilized MTs. Since saponin, irrespective of subsequent extraction times, always produced a Glu-positive reaction and ultrastructural analysis never gave compelling evidence for a drastic MAP-removal, we conclude that the Glu-epitope became freely accessible due to conformational changes in the tubulin polymeres.
Mütze
W
W
Ruhr-Universität Bochum, Lehrstuhl für Zellmorphologie, Bochum, Germany.
Breuker
R N
RN
Hauser
M
M
D000818:Animals; D000911:Antibodies, Monoclonal; D003902:Detergents; D016822:Euglenida / Q000378:metabolism* / Q000648:ultrastructure; D005247:Feeding Behavior; D005455:Fluorescent Antibody Technique; D018698:Glutamic Acid / Q000737:chemistry / Q000378:metabolism*; D008854:Microscopy, Electron; D015388:Organelles / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D014404:Tubulin / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D014443:Tyrosine / Q000737:chemistry / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D003902:Detergents; D014404:Tubulin; D018698:Glutamic Acid; D014443:Tyrosine