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Analysis of Rab42 and Rab40c as novel regulators of secretory membrane trafficking

Tsai, Yueh-Tso

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Secretory membrane trafficking is essential for the homeostasis of most cellular organelles and mediating the secretion of a variety of cargo proteins, including digestive enzymes, hormones, growth factors, antibodies, extracellular matrix proteins and many other proteins. Small GTPases, particularly Rab and Arf GTPases, have been shown to play key roles in regulating the transport of secretory cargo proteins. Nevertheless, the biological role of each individual small GTPase is not fully elucidated yet. To address that, I performed microscopy-based RNAi screening to identify small GTPases that are involved in the biosynthetic transport of a secretory cargo protein ts-O45-G. From RNAi screen in HeLa cells, I identified 18 potential regulators, with 14 of them not associated with this cellular function before. One of the hits was chosen for further characterization. Rab42 is an interesting hit in that it has not been characterized and the retroposed transcript of Rab42 is expressed in HeLa cells. After having designed specific siRNAs only targeting Rab42, it is shown that specific down-regulation of Rab42 inhibited secretion of ts-O45-G from the ER to the PM. Moreover, I showed that RNAi-mediated inhibition of ts-O45-G transport can be rescued by over-expressing a siRNA-resistant form of Rab42. By invesitigating more closely which particular stage of ts-O45-G secretion is affected, I have identified that trafficking from the TGN to the PM is affected in a more pronounced manner. Immunofluorescence analysis showed that over-expressed Rab42 localized preferentially to the cytoplasm and PM, and, to a lesser extent, to the intracellular punctual structures, which are in close proximity to perinuclear region. During my thesis work, yet another interesting and poorly characterized Rab, Rab40c, was identified as a novel regulator for PC I secretion from our RNAi screens in NIH3T3 fibroblasts. Down-regulation of Rab40c inhibited secretion of PC I and ts-O45-G in NIH3T3 fibroblasts, albeit it had no consistent effect on ts-O45-G secretion in HeLa cells. I showed that PC I secretion is blocked in the early secretory pathway in NIH3T3 fibroblasts depleted for Rab40c. In addition, I observed cell type-specific localization of Rab40c by comparing the localization of Rab40c in NIH3T3 fibroblasts and HeLa cells. In NIH3T3 fibroblasts FLAG-tagged Rab40c localized to the Golgi complex, whereas in HeLa cells FLAG-tagged Rab40c localized to perinuclear tubulovesicular structures and cytoplasmic structures. Furthermore, Rab40c is an interesting Rab GTPase in that it contains a GTPase domain and a SOCS-box domain. By immunofluorescence analysis, it has been shown that SOCS-box domain is crucial for the localization of Rab40c at the Golgi complex. Taken together, my thesis work has identified novel regulators of secretory membrane trafficking, and serves as a basis for further functional characterization.

Item Type: Dissertation
Supervisor: Nickel, Prof. Dr. Walter
Date of thesis defense: 2 October 2013
Date Deposited: 22 Oct 2013 06:24
Date: 2013
Faculties / Institutes: Service facilities > Bioquant
Subjects: 500 Natural sciences and mathematics
570 Life sciences
610 Medical sciences Medicine
Controlled Keywords: Rab GTPases, secretory pathway, ts-O45-G
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