ANTEROGRADE AND RETROGRADE TRAFFIC BETWEEN THE ROUGH ENDOPLASMIC-RETICULUM AND THE GOLGI-COMPLEX

The transfer of newly synthesized membrane proteins moving from the ro ugh endoplasmic reticulum (RER) to the Golgi complex has been studied by electron microscopy in HEp-2 cells transfected with cDNAs for chime ric proteins, These proteins consist of a reporter enzyme, horseradish peroxidase (HRP), anchored to the transmembrane domains of two integr al membrane proteins, the transferrin receptor and sialyltransferase. The chimeras are distributed throughout the nuclear envelope, RER, ves icular tubular clusters (VTCs) and a network of tubules in the cis-Gol gi area. At 20 degrees C tubules containing chimera connect the RER to the VTCs and to the cis-Golgi network. On transfer to 37 degrees C in the presence of dithiothreitol (DTT), the chimeras are seen to move f rom the RER and through the Golgi stack, With this temperature shift t he direct connections with the RER are lost and free vesicles form; so me of these vesicles contain HRP reaction product which is much more c oncentrated than in the adjacent RER while others lack reaction produc t entirely. In cells expressing SSHRP(KDEL), DAB reaction product rema ins distributed throughout the RER, the VTCs, and the cis-Golgi networ k for prolonged periods in the presence of DTT and almost all of the v esicles which form at 37 degrees C are DAB-positive. Together these ob servations demonstrate that all three chimeras are transported from th e RER to the cis-Golgi in free, 40-60-nm vesicles at 37 degrees C, The y also suggest that the retrograde traffic which carries SSHRP(KDEL) b ack to the RER is probably mediated by vesicles with a similar morphol ogy but which, in cells expressing membrane-anchored chimeras, lack de tectable reaction product.