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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...

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Formato: Texto
Lenguaje:English
Publicado: The Rockefeller University Press 1994
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2120231/
https://www.ncbi.nlm.nih.gov/pubmed/7962049
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description 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.
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spelling pubmed-21202312008-05-01 Transport into and out of the Golgi complex studied by transfecting cells with cDNAs encoding horseradish peroxidase J Cell Biol Articles 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. The Rockefeller University Press 1994-11-01 /pmc/articles/PMC2120231/ /pubmed/7962049 Text en This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 4.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/4.0/).
spellingShingle Articles
Transport into and out of the Golgi complex studied by transfecting cells with cDNAs encoding horseradish peroxidase
title Transport into and out of the Golgi complex studied by transfecting cells with cDNAs encoding horseradish peroxidase
title_full Transport into and out of the Golgi complex studied by transfecting cells with cDNAs encoding horseradish peroxidase
title_fullStr Transport into and out of the Golgi complex studied by transfecting cells with cDNAs encoding horseradish peroxidase
title_full_unstemmed Transport into and out of the Golgi complex studied by transfecting cells with cDNAs encoding horseradish peroxidase
title_short Transport into and out of the Golgi complex studied by transfecting cells with cDNAs encoding horseradish peroxidase
title_sort transport into and out of the golgi complex studied by transfecting cells with cdnas encoding horseradish peroxidase
topic Articles
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2120231/
https://www.ncbi.nlm.nih.gov/pubmed/7962049