DIRECT PATHWAY FROM EARLY RECYCLING ENDOSOMES TO THE GOLGI-APPARATUS REVEALED THROUGH THE STUDY OF SHIGA TOXIN B-FRAGMENT TRANSPORT/

Shiga toxin and other toxins of this family can escape the endocytic p athway and reach the Golgi apparatus. To synchronize endosome to Golgi transport, Shiga toxin B-fragment was internalized into HeLa cells at low temperatures. Under these conditions, the protein partitioned awa y from markers destined for the late endocytic pathway and colocalized extensively with cointernalized transferrin. Upon subsequent incubati on at 37 degrees C, ultrastructural. studies on cryosections failed to detect B-fragment-specific label in multivesicular or multilamellar l ate endosomes, suggesting that the protein bypassed the late endocytic pathway on its way to the Golgi apparatus. This hypothesis was furthe r supported by the rapid kinetics of B-fragment transport, as determin ed by quantitative confocal microscopy on living cells and by B-fragme nt sulfation analysis, and by the observation that actin-depolymerizin g and pH-neutralizing drugs that modulate vesicular transport in the l ate endocytic pathway had no effect on B-fragment accumulation in the Golgi apparatus. B-fragment sorting at the level of early/recycling en dosomes seemed to involve vesicular coats, since brefeldin A treatment led to B-fragment accumulation in transferrin receptor-containing mem brane tubules, and since B-fragment colocalized with adaptor protein t ype 1 clathrin coat components on early/recycling endosomes. Thus, we hypothesize that Shiga toxin B-fragment is transported directly from e arly/recycIing endosomes to the Golgi apparatus. This pathway may also be used by cellular proteins, as deduced from our finding that TGN38 colocalized with the B-fragment on its transport from the plasma membr ane to the TGN.