Distinct caveolae-mediated endocytic pathways target the Golgi apparatus and the endoplasmic reticulum

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Abstract
Internalization of autocrine motility factor (AMF) into the endoplasmic reticulum is sensitive to the cholesterol-extracting reagent methyl-β-cyclodextrin, inhibited by the dynamin-1 K44A mutant and negatively regulated by caveolin-1. Thus, AMF internalization requires a caveolae-mediated endocytic pathway. Similarly, we show here that endocytosis of cholera toxin (CTX) in NIH-3T3 fibroblasts is inhibited by adenoviral expression of the dynamin-1 K44A mutant but only partially by expression of the clathrin hub. Treatment with methyl-β-cyclodextrin and overexpression of caveolin-1, but not the clathrin hub, selectively diminishes CTX endocytosis to the Golgi apparatus but not to endosomes. CTX is therefore targeted via a caveolin-1-regulated caveolae-mediated pathway to the Golgi. Disruption of Golgi-, caveosome- or endosome-mediated trafficking with brefeldin A, nocodazole or a 20°C temperature block, respectively, inhibit CTX endocytosis to the Golgi but do not affect AMF delivery to the endoplasmic reticulum. Following an incubation of only five minutes in the presence of the clathrin hub, AMF and CTX are not cointernalized, and AMF is delivered to the AMF-R-positive smooth ER. The internalization of both ligands is nevertheless sensitive to the tyrosine kinase inhibitor genistein, confirming that they are both internalized via caveolae/raft pathways. Two distinct caveolae-mediated endocytic pathways therefore exist, including a novel direct pathway to the ER from the plasma membrane.