8 research outputs found
Eeyarestatin 1 interferes with both retrograde and anterograde intracellular trafficking pathways
Background: The small molecule Eeyarestatin I (ESI) inhibits the endoplasmic reticulum (ER)-cytosol dislocation and
subsequent degradation of ERAD (ER associated protein degradation) substrates. Toxins such as ricin and Shiga/Shiga-like
toxins (SLTx) are endocytosed and trafficked to the ER. Their catalytic subunits are thought to utilise ERAD-like mechanisms
to dislocate from the ER into the cytosol, where a proportion uncouples from the ERAD process, recovers a catalytic
conformation and destroys their cellular targets. We therefore investigated ESI as a potential inhibitor of toxin dislocation.
Methodology and Principal Findings: Using cytotoxicity measurements, we found no role for ESI as an inhibitor of toxin
dislocation from the ER, but instead found that for SLTx, ESI treatment of cells was protective by reducing the rate of toxin
delivery to the ER. Microscopy of the trafficking of labelled SLTx and its B chain (lacking the toxic A chain) showed a delay in
its accumulation at a peri-nuclear location, confirmed to be the Golgi by examination of SLTx B chain metabolically labelled
in the trans-Golgi cisternae. The drug also reduced the rate of endosomal trafficking of diphtheria toxin, which enters the
cytosol from acidified endosomes, and delayed the Golgi-specific glycan modifications and eventual plasma membrane
appearance of tsO45 VSV-G protein, a classical marker for anterograde trafficking.
Conclusions and Significance: ESI acts on one or more components that function during vesicular transport, whilst at least
one retrograde trafficking pathway, that of ricin, remains unperturbed
Diacylglycerol kinase and phospholipase D inhibitors alter the cellular lipidome and endosomal sorting towards the Golgi apparatus
Phosphoinositides in the Mammalian Endo-lysosomal Network
The endo-lysosomal system is an interconnected tubulo-vesicular network that acts as a sorting station to process and distribute internalised cargo. This network accepts cargoes from both the plasma membrane and the biosynthetic pathway, and directs these cargos either towards the lysosome for degradation, the peri-nuclear recycling endosome for return to the cell surface, or to the trans-Golgi network. These intracellular membranes are variously enriched in different phosphoinositides that help to shape compartmental identity. These lipids act to localise a number of phosphoinositide-binding proteins that function as sorting machineries to regulate endosomal cargo sorting. Herein we discuss regulation of these machineries by phosphoinositides and explore how phosphoinositide-switching contributes toward sorting decisions made at this platform