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Molecular cloning, localization and functional studies of Rab18, a low molecular weight GTP-binding protein from mouse pituitary cells

Posted on:1997-05-19Degree:Ph.DType:Dissertation
University:University of California, BerkeleyCandidate:Yu, HelenFull Text:PDF
GTID:1464390014981092Subject:Biology
Abstract/Summary:
The Rab family of Ras-related low molecular weight GTP-binding proteins has been implicated to play a role in controlling vesicle trafficking in eukaryotic cells from yeast to mammals. To search for novel Rabs associated with constitutive as well as specialized secretory or endocytic pathways in mammalian cells, I used a PCR-based approach to amplify Rab effector domains from mouse pituitary cells. The full length gene encoding a novel Rab protein, Rab18, was subsequently isolated from a cDNA library of the mouse pituitary cell line AtT20. Analysis of the predicted amino acid sequence of Rab18 indicates that it is a bona fide Rab. Recombinant Rab18 also functionally binds GTP. Tissue survey of Rab18 expression by both northern and western analysis show that Rab18 is present in all tissues but is highly expressed in the brain. Velocity and equilibrium gradient fractionation of PC12 cells suggest that Rab18 is present on a population of membrane vesicles with similar size and density as synaptic-like micro-vesicles (SLMV). However, immuno-adsorption of SLMV using anti-synaptophysin antibodies indicates that Rab18 is excluded from these vesicles. Immuno-electron microscopy of Rab18 in rat brain synaptosomes shows that Rab18 is present on micro-vesicles 40-60 nm in diameter but is not found on synaptophysin containing synaptic vesicles, consistent with results from PC12 cell fractionation and SLMV immuno-adsorption studies. Immuno-localization of Rab18 in transfected CHO cells shows partial overlap of the protein with the fluid phase endocytic tracer BSA-Rh but not with transferrin-Rh. Overexpression of wildtype Rab18 enhanced BSA-Rh uptake but inhibited transferrin-Rh and transferrin-HXP internalization. This inhibitory effect of wildtype Rab18 can be "rescued" by co-expression of the dominant negative form of Rab18. Thus, Rab18 may be involved in an endocytic pathway that is in parallel with the clathrin-dependent pathway utilized by transferrin. Rab18 was also found to redistribute from cytosol to membranes following stimulation with high K...
Keywords/Search Tags:Rab18, Mouse pituitary, Molecular, Protein, Cells
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