| Summary: | Vesicle trafficking plays an important role in delivering a diverse range of cargoes between different membranous systems in eukaryotes. It is well documented that the brefeldin A (BFA)-inhibited guanine nucleotide exchange factor (GEF), named BIG, regulates vesicle budding at the <i>trans-</i>Golgi network (TGN) and recycling endosomes through activating the ADP-ribosylation factor (ARFs). Among the five BIGs in <i>Arabidopsis</i>, BIG5 is characterized to mediate ARF-dependent trafficking at the plasma membrane or endosomes while the members from BIG1 to BIG4 (BIG1-BIG4) at the TGN in the secretory pathway. However, evidence is increasing to suggest that BIG5 can function redundantly with BIG1-BIG4 to regulate vesicular trafficking in response to various intra- and extra-cellular stimuli. In this study, our genetic analysis showed that BIG5 played an overlapping role at least with BIG3 in cell proliferation. To elucidate molecular mechanisms underlying the BIG5- and BIG3-regulated biological processes, we examined the effect of BIGs on expression patterns of the two transmembrane proteins, PINFORMED 2 (PIN2) epically localized in root epidermal cells and the regulator of G protein signaling 1 (RGS1) localized in the plasma membrane. Our data showed that the PIN2 polar distribution was slightly reduced in <i>big3 big5</i> in the absence of BFA, and it was significantly reduced by the treatment of 0.1 µM BFA in <i>big3 big5</i>. Further analysis revealed that BFA bodies derived from the plasma membrane were only observed in wild type (WT), <i>big3</i> and <i>big5</i> cells, but not in the <i>big3 big5</i> cells. These results indicate that BIG5 and BIG3 are functionally redundant in the endosome recycling pathway from the plasma membrane to TGN. On the other hand, the single <i>BIG3</i> or <i>BIG5</i> mutation had no effect on the plasma membrane expression of RGS1, whereas the double mutations in <i>BIG3</i> and <i>BIG5</i> led to a significant amount of RGS1 retained in the vesicle, indicating that BIG3 and BIG5 act redundantly in mediating protein trafficking. Furthermore, transmission electron microscopy assays showed that Golgi ultrastructure in <i>big3 big5</i> cells was abnormal and similar to that in BFA-treated WT cells. Taken together, our data provide several new lines of evidence supporting that BIGs play a redundant role in vesicular trafficking and probably also in maintaining the Golgi structural integrity in <i>Arabidopsis</i>.
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