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Targeting of membrane proteins to lysosome-related organelles

Kaur, Jasber; (2003) Targeting of membrane proteins to lysosome-related organelles. Doctoral thesis (Ph.D), UCL (University College London). Green open access

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Lysosome-related organelles are cellular compartments that sequester secretory and lysosomal components. A number of different genetic disorders, such as Chediak-Higashi Syndrome, Hermansky-Pudlak Syndrome and Griscelli Syndrome, display abnormal cellular phenotypes predominantly in cells with lysosome-related organelles. Some of the causative mutated genes in these disorders encode ubiquitously expressed components predicted to mediate vesicular traffic to conventional lysosomes as well as lysosome-related organelles. In this thesis, the question of how membrane proteins are sorted to lysosome-related organelles is addressed. The adhesion receptor P-selectin and the tetraspanin CD63, which both localise to lysosome-related organelles, have been used to determine the targeting requirements for sorting to this compartment. Rbl-2H3 cells possess a secretory organelle that is lysosome-related, but no separate conventional lysosome pool. These cells were used to determine the sorting signal requirements for localisation of horseradish peroxidase (HRP)-P-selectin chimeras within Rbl-2H3 lysosome-related organelles. Subcellular fractionation, immunofluorescence microscopy, and the monitor of proteolysis of HRP-P-selectin chimeras, reveal that HRP-P-selectin uses the same targeting sequences for delivery to the Rbl-2H3 lysosome-related organelle as for delivery to conventional lysosomes. Human Umbilical Vein Endothelial Cells (HUVEC) were used to investigate the sequence requirements for sorting of CD63 to both Weibel-Palade bodies (WPB), which are lysosome-related, and to conventional lysosomes. Mutants with altered abilities to bind AP3 and AP2, both components of the cytoplasmic sorting machinery, were constructed as GFP-CD63 chimeras and expressed in HUVEC. Microscopical analysis of their targeting to both WPB and conventional lysosomes revealed that AP3 is more important in traffic of GFP-CD63 mutants to WPB than to conventional lysosomes. This thesis supports the hypothesis that conventional lysosome trafficking pathways are enhanced to mediate the specific transport of components to lysosome-related organelles, whether they coexist with conventional lysosomes or not.

Type: Thesis (Doctoral)
Qualification: Ph.D
Title: Targeting of membrane proteins to lysosome-related organelles
Open access status: An open access version is available from UCL Discovery
Language: English
Additional information: Thesis digitised by ProQuest.
Keywords: Biological sciences; Lysosome-related organelles
URI: https://discovery.ucl.ac.uk/id/eprint/10100884
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