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Title: Reconstitution of TGN to endosome transport in vitro
Author: Cook, N. R.
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2002
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The trans-Golgi network (TGN) is one of the major intracellular membrane sorting stations in the cell. Here a number of specific proteins are segregated from the pathway of constitutive secretion and transported to a variety of membrane bound organelles. It is thought that newly synthesised lysosomal components are sorted here for delivery to the endocytic pathway. Examples of such proteins are a number of highly glycosylated integral membrane proteins enriched in the limiting membrane of lysosomes. The majority of these proteins are believed to reach this compartment by direct transport from the TGN to endosomes without prior delivery to the cell surface. However the precise membrane trafficking events and the molecular machinery governing this process are currently poorly understood. To get new insight into transport from the TGN to endosomes I focused upon the integral membrane protein lysosomal glycoprotein 120 (lsp120). Tyrosine sulphation has been used in a number of previous studies to specifically label proteins in the TGN. Thus to more closely define the trafficking of lgp120 I created chimeras of this protein incorporating potential tyrosine sulphation motifs and introduced a lumenally oriented modified chicken avidin molecule to enable collection of the synthetic protein in endosomes. Stable expression of this chimera in HeLa cells demonstrated that it was efficiently delivered to lysosomes and this was mediated by the tyrosine based signal in its cytoplasmic tail. The chimera could be specifically labelled with radioactive sulphate and bound biotin with high affinity. This formed the basis of novel assays to measure transport from the TGN to endosomes both in the intact cell and in vitro. In each instance delivery to endosomes was determined by binding of the sulphated chimera to internalised biotinylated immunoglobulin in sealed membrane bound compartments. This new approach indicated that newly synthesised lysosomal membrane proteins are delivered directly to early endosomes.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available