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PMID: 7962049 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't

Transport into and out of the Golgi complex studied by transfecting cells with cDNAs encoding horseradish peroxidase.

The Journal of cell biology ·Vol. 127 ·No. 3 ·1994-11-00 ·Pages 641-52

Connolly CN, Futter CE, Gibson A, Hopkins CR, Cutler DF

Abstract

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.

MeSH Terms
Animals Base Sequence Carcinoma, Hepatocellular Cell Line Chlorocebus aethiops DNA Primers DNA, Complementary Endocytosis Endoplasmic Reticulum/metabolism,ultrastructure Endosomes/metabolism Genes, myc Golgi Apparatus/metabolism,ultrastructure Horseradish Peroxidase/biosynthesis,metabolism Humans Kidney Kinetics Liver Neoplasms Molecular Sequence Data Polymerase Chain Reaction Receptors, Transferrin/analysis,metabolism Transfection
Chemicals
DNA Primers DNA, Complementary Receptors, Transferrin Horseradish Peroxidase
Authors & Affiliations
5 authors, click to expand affiliations / ORCID
Connolly C N
Medical Research Council Laboratory for Molecular Cell Biology, University College London, United Kingdom.
Futter C E
Gibson A
Hopkins C R
Cutler D F
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Article Info
Journal
The Journal of cell biology
Abbr.
J Cell Biol
ISSN
0021-9525
DOI
10.1083/jcb.127.3.641
Published
1994-11-00
Pages
641-52
Language
English
Region
United States
NLM ID
0375356
PMCID
PMC2120231
Subset
IM
External Links
PubMed source DOI: 10.1083/jcb.127.3.641 PMC Full Text View journal details
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