Dynamics and distribution of ARFB1b and ARFB1c GTPases in N. <i>tabacum</i> plant cells

Abstract

Eukaryotic cells are characterized by a complex of endomembranes. The main organelles of this complex are the endoplasmic reticulum, the Golgi apparatus, the trans-Golgi network, the vacuoles, and the plasma membrane. In cells the endomembrane system identity and interconnection is preserved by an active intracellular trafficking mediated by vesicles, which shuttle cargo molecules such as proteins, polysaccharides, and lipids between these organelles. During the intracellular trafficking, vesicle fusion and budding is driven by the assembly and disassembly of the coat proteins from the membrane. In turn, assembly and disassembly of coat proteins are regulated by other proteins called ADP-Ribosylation Factors (ARFs), a subfamily of the Ras GTP-binding proteins superfamily, which switching from the inactive form (ARFGDP) to the active form (ARFGTP) regulates the assembly and the disassembly of the coat proteins. The conversion between the inactive and active form is mediated by Guanine Exchange Factors (GEFs), while the active form is converted into the inactivated form by GTPase Activating Proteins (GAPs). ARFGTP peripherally associates with the organelle membrane and starts to recruits coat proteins from the cytosol. These coat proteins drive the membrane budding, capturing at the same time specific cargo molecules. After the hydrolysis of the ARFGTP form into ARFGDP, which is released into the cytosol, the coat component proteins detach from the membrane. In this way vesicles can dock and fuse with the target membrane. <br /> In yeast and mammalian cells an active role of ARFs in the endocytic pathway has been demonstrated by creating mutants of this protein and seeing a blockage of this pathway. Vesicle trafficking in plants has functions similar to those in animal and yeast. In addition, it is necessary for non-cellulose polysaccharides delivery to the plant cell wall. <br /> All the regulatory roles in which ARFs are involved: membrane traffic, lipid metabolism, organelle morphology and cellular signalling, have been difficult to dissect due to the complexity of the ARF family. <br /> In A. thaliana 12 ARFs have been identified, but for most of them the functionality is completely unknown. Moreover, no role has yet been identified for any of them, in the endocytic pathway. <br /> Bioinformatics analysis of ARF proteins: In this work, the selection of possible ARFs involved in the endocytic pathways has been done making a BlastP of A. thaliana ARFs sub-family, considering the presence of the specific motif MxxE, which enables ARF1 to localize to the early Golgi apparatus. Recent evidence has shown that the motifs MxxE match the motif ILTD in ARFB1a (the homologue of the human ARF6). For this protein, the motif ILTD enables ARFB1a to localize at the plasma membrane and to take part in the endocytic pathway. Using BlastP other two ARFs were identified: ARFB1b and ARFB1c with a similar motif IIKD and IIRD and with an additional DPF motif important for AP2 interaction in human. All these characteristics suggest these two proteins as ideal candidate for having crucial function in the endocytotic machinery. ArfB1b and ArfB1c localization: For each of these proteins GFP/YFP fusions were created. The constructs so obtained were used to localize them in living epidermis cells of Nicotiana tabacum by confocal microscopy. The novel proteins plus ARF1 and ARFB1a (used as controls) were coexpressed with ERD2, a marker for the Golgi apparatus, with the SNARE SYP61, a marker for the Trans Golgi network, with the FYVE domain construct, marker for the early and late endosomes and with the two RAB GTPases RHAIand ARA7 markers of the PVC (pre-vacuolar compartment) and the early and late endosomes respectively. The experiment of colocalization with all these specific markers localizes both ARFB1b and ARFB1c at the TGN (Trans Golgi network) and for ARFB1c a partial co-localization with FYVE domain construct and ARA7 was observed. Furthermore cross-colocalization between ARF1, ARFB1a, ARFB1b and ARFB1c, shows that these proteins overlap in their colocalization on the trans Golgi apparatus. Kinetic analysis: To evaluate the cellular dynamics of the ARF proteins used in this work, a study of the kinetics of these proteins was performed using FRAP (Fluorescence Recovery After Photobleaching) analysis. <br /> A different kinetic was found for each of the examined proteins. <br /> ARF1 wt has a relatively slow half maximal recovery time equal to 10.9± 1.5 s (seconds), compared to the wt form ARF1GTP which has a recovery time of 19± 3.0 s, ARFB1awt recovers in 16± 1.4 s, and ARFB1aGTP which recovers in 33.6± 3.2 s. ARFB1bwt recovers in 12.2± 2.6 s and the GTP bound form in 16.3± 2.8 s; for ARFB1c the half maximal recovery time is 12.6± 1.5 s, while its GTP form recycles in 24.3± 4.2 s. <br /> The fluorescence recovery time of some ARFGTPs at the Golgi or non Golgi apparatus membrane suggested that the GTP mutants’ ability recycling is not entirely abolished, the kinetic studies also show that ARFs are highly mobile and that ARFs GTP bound form diffuses more slowly compared to ARFs in wt form. Furthermore, the different recovery times suggest a distinct role in the secretory pathway. The higher speed of recovery for ARF1 and ARF1GTP compared to the ARFB1a, which localizes to the plasma membrane, suggests that this protein is important to maintain equilibrium between the anterograde and the retrograde pathways. Similar observations for the ARFB1b and ARFB1c, suggest that they may maintain equilibrium during the traffic at the TGN, which involves endosomes, plasma membrane and PVC. <br /> ARF1, ARFB1a, ARFB1b and ARFB1c have different impacts on protein secretion in N. tabacum transformed protoplast: To evaluate the impact of these ARFs on the protein secretion the wt forms of the proteins were co-transformed with the secretory marker SecRGUS. The results obtained were compared with another experiment in which the impact on the secretion of the GDP mutant form was evaluated. The secretion test shows that ARF1GDP inhibits proteins secretion. ARFB1bwt stimulates secretion, compared to ARF1wt and ARFB1a. In contrast ARFB1bGDP hampers secretion more than ARF1GDP. ARFB1c has more or less no effect on the secretion in the wt form, while in the GDP form it seems to have less inhibitory effect than ARFB1bGDP. Taking together these results suggest that ARFB1b and ARFB1c have a remarkable influence on secretion processes. Furthermore it is hypothesized that they are involved in two different secretory pathways, ARFB1b directed to the plasma membrane and ARFB1c to the endosomes. These results show that blocking the pathway in which one of the proteins is involved, it is stimulated a parallel pathway probably to maintain stability and structure of the endomembrane system, in order to protect the equilibrium and functionality of the cell.