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 morphological basis of exocytotic traffic. We have used expression of HRP from cDNA in a variety of cells in combination with peroxidase cytochemistry to outline traffic into and out of the Golgi apparatus at the electron microscopic level with very high sensitivity. A secretory form of the peroxidase (ssHRP) is active from the beginning of the secretory pathway and the activity is efficiently cleared from cells. Investigation of the morphological elements involved in the itinerary of soluble ER proteins using ssHRP tagged with the ER retention motif (ssHRPKDEL) shows that it progresses through the Golgi stack no further than the cis-most element. Traffic between the RER and the Golgi stack as outlined by ssHRPKDEL occurs via vesicular carriers as well as by tubular elements. ssHRP has also been used to investigate the trans side of the Golgi complex, where incubation at reduced temperatures outlines the trans-Golgi network with HRP reaction product. Tracing the endosomal compartment with transferrin receptor in double-labeling experiments with ssHRP fails to show any overlap between these two compartments.
Medical Research Council Laboratory for Molecular Cell Biology, University College London, United Kingdom.
C N
CN
Connolly
C E
CE
Futter
A
A
Gibson
C R
CR
Hopkins
D F
DF
Cutler
D000818:Animals; D001483:Base Sequence; D006528:Carcinoma, Hepatocellular; D002460:Cell Line; D002522:Chlorocebus aethiops; D017931:DNA Primers; D018076:DNA, Complementary; D004705:Endocytosis; D004721:Endoplasmic Reticulum / Q000378:metabolism / Q000648:ultrastructure; D011992:Endosomes / Q000378:metabolism; D016259:Genes, myc; D006056:Golgi Apparatus / Q000378:metabolism* / Q000648:ultrastructure; D006735:Horseradish Peroxidase / Q000096:biosynthesis / Q000378:metabolism*; D006801:Humans; D007668:Kidney; D007700:Kinetics; D008113:Liver Neoplasms; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011990:Receptors, Transferrin / Q000032:analysis / Q000378:metabolism; D014162:Transfection
D017931:DNA Primers; D018076:DNA, Complementary; D011990:Receptors, Transferrin; D006735:Horseradish Peroxidase