Functional and Kinetic studies of COPI machinery

Abstract

Abstract In this study, different aspects of vesicular transport in the early secretory pathway have been investigated. Although an earlier study showed an effect upon the down regulation of β-COP in the transport of the SH4 domain containing HASPB and Yes (Ritzerfeld et al., 2011), there was no study revealing the role of coatomer subunits in plasma membrane localization of unconventionally secreted proteins. This work investigates the role of γ2-COP in the transport of the unconventionally secreted proteins HASPB and Yes. Pronounced retention of HASPB at the perinuclear region was observed after down regulation of the coatomer subunits α, β, δ, and ζ suggesting that the coatomer subunits and therefore most likely COPI vesicles are involved in the transport of HASPB. However individual knockdowns of γ1- and γ2-COP subunits did not affect the transport of the reporter proteins, indicating there is no specific role of coatomer isoforms per se. In the second part, the impact of novel Brefeldin A (BFA) analogs on the Golgi morphology was investigated. Treatments of mammalian cells with various analogues revealed that number of derivatives had no impact on the Golgi morphology in given times in addition to some derivatives causing reversible Golgi disruption as Brefeldin A. The variation of the effect can be of use in various fields for creating new therapeutics. Another part of this work focusing on the oligomerization of the p24 protein and a non-SM18 binding p24 variant (L17F) were studied by cross-linking the proteins in vivo using a chemical cross-linking agent. This shows a significant reduction in the oligomeric form of the non-SM18 binding p24 variant compared to the monomeric form. The final part of this thesis presents the findings on the kinetics of p24 family members using Fluorescent Loss in Photobleaching (FLIP). Experiments resulted in no significant differences between transport rates of p23 and p24 from the Golgi to the endoplasmic reticulum.