The predominant secretory cargo of
bloodstream form
Trypanosoma brucei
is Variant
Surface Glycoprotein (VSG), comprising ~10% total
protein and forming a dense protective layer.
Blocking VSG translation using Morpholino
oligonucleotides triggered a precise pre/cytokinesis
arrest. We investigated the effect of blocking VSG
synthesis on the secretory pathway. The number of
Golgi decreased, particularly in post/mitotic cells,
from 3.5 ± 0.6 to 2.0 ± 0.04 per cell. Similarly, the
number of ER exit sites (ERES) in post/mitotic cells
dropped from (3.9 ± 0.6) to (2.7 ± 0.1) eight hours
after blocking VSG synthesis. The secretory
pathway was still functional in these stalled cells, as
monitored using Cathepsin L. Rates of
phospholipid and GPI/anchor biosynthesis were
relatively unaffected, except for the level of
sphingomyelin which increased. However, both ER
and Golgi morphology became distorted, with the
Golgi cisternae becoming significantly dilated,
particularly at the trans/face. Membrane
accumulation in these structures is possibly caused
by reduced budding of nascent vesicles due to the
drastic reduction in the total amount of secretory
cargo, i.e. VSG. These data argue that the total flux
of secretory cargo impacts upon the biogenesis and
maintenance of secretory structures and organelles
in
T. brucei
including the ERES and Golgi.