The small GTPase RAB24 and vesicle trafficking from the endoplasmic reticulum to the Golgi apparatus

Abstract

The small GTPase RAB24 is an unusual RAB protein, which is originally reported to localize in the endoplasmic reticulum (ER), cis-Golgi region and late endosomal structures. Several studies have shown that RAB24 is involved in autophagy, endocytosis and cell division, but its association to the cis-Golgi has not been studied. However, preliminary data from the host laboratory implied that RAB24 may play a role in ER-Golgi trafficking. Therefore, the aims of this thesis were to study the structural integrity and location of the Golgi complex in wild-type (WT) and RAB24-knockout (KO) hepatocyte cell lines, and to investigate the role of RAB24 in the ER-Golgi vesicle transport. The integrity of the Golgi ribbons was analyzed by culturing WT and RAB24-KO Huh7 cells at +37°C and +40°C, and staining the cells using an antibody against the Golgi marker GM130. A GFP-tagged, temperature-sensitive vesicular stomatitis virus glycoprotein (VSVG-ts045-eGFP) was used to conduct the ER-Golgi protein trafficking assay. At +40 °C, VSVG-ts045-eGFP protein cannot exit the ER due to misfolding. When the cells are shifted to +32 °C, the synchronized transport of the protein from the ER to the Golgi can be monitored. For the vesicle trafficking assay, cells expressing VSVG-ts045-eGFP were collected at different time points after shifting from +40 °C to +32 °C, and the colocalization of eGFP and the Golgi marker GM130 were analyzed using confocal microscopy. Further, deglycosylation assay was performed using endoglycosidase H (Endo H) enzyme that removes N-linked glycans from proteins that have not exited the ER. Western blotting was used to analyze the presence of VSVG-ts045-eGFP protein at the ER and post-ER compartments in WT and RAB24-KO cells at different time points after shifting the cells from +40°C to +32°C. At +37 °C, the Golgi ribbons were positioned on top of the nucleus in WT cells, whereas in the RAB24-KO cells, the Golgi ribbons localized around the nucleus. At +40 °C, the GM130-positive Golgi ribbons were fragmented in both WT and RAB24-KO cells, likely due to heat stress. Confocal microscopy revealed retarded ER-to-Golgi trafficking of VSVG-ts045-eGFP in RAB24-KO cells compared with the WT cells. However, no differences were observed in the Endo H sensitivity assay between the two cell lines. The results suggest that RAB24 may play a role in positioning of the Golgi apparatus in relation to the nucleus, and in ER-to-cis-Golgi trafficking.