TRANSPORT INTO AND OUT OF THE GOLGI-COMPLEX STUDIED BY TRANSFECTING CELLS WITH CDNAS ENCODING HORSERADISH-PEROXIDASE

We have developed a novel technique with which to investigate the morp hological basis of exocytotic traffic. We have used expression of HRP from cDNA in a variety of cells in combination with peroxidase cytoche mistry to outline traffic into and out of the Golgi apparatus at the e lectron microscopic level with very high sensitivity. A secretory form of the peroxidase (ssHRP) is active from the beginning of the secreto ry pathway and the activity is efficiently cleared from cells. Investi gation of the morphological elements involved in the itinerary of solu ble ER proteins using ssHRP tagged with the ER retention motif (ssHRP( KDEL)) shows that it progresses through the Golgi stack no further tha n the cis-most element. Traffic between the RER and the Golgi stack as outlined by ssHRP(KDEL) occurs via vesicular carriers as well as by t ubular elements. ssHRP has also been used to investigate the trans sid e of the Golgi complex, where incubation at reduced temperatures outli nes the trans-Golgi network with HRP reaction product. Tracing the end osomal compartment with transferrin receptor in double-labeling experi ments with ssHRP fails to show any overlap between these two compartme nts.