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Peripheral tethering factors bind to small GTPases in order to obtain their correct location within the Golgi apparatus. Using fluorescence resonance energy transfer (FRET) and fluorescence lifetime imaging microscopy (FLIM) we visualized interactions between Arabidopsis homologues of tethering factors and small GTPases at the Golgi stacks in planta. Co-expression of the coiled-coil proteins AtGRIP and golgin candidate 5 (GC5) [TATA element modulatory factor (TMF)] and the putative post-Golgi tethering factor AtVPS52 fused to green fluorescent protein (GFP) with mRFP (monomeric red fluorescent protein) fusions to the small GTPases AtRab-H1b, AtRab-H1c and AtARL1 resulted in reduced GFP lifetimes compared to the control proteins. Interestingly, we observed differences in GFP quenching between the different protein combinations as well as selective quenching of GFP-AtVPS52-labelled structures. The data presented here indicate that the FRET-FLIM technique should prove invaluable in assessing protein interactions in living plant cells at the organelle level.
The Golgi apparatus plays a central role in the plant endomembrane system, as it is not only involved in the processing of protein cargo received from the endoplasmic reticulum (ER) but also in the synthesis of complex cell wall polysaccharides (1). Plant cells can contain up to hundreds of discrete Golgi stacks that are mobile in an actin-dependent manner with the surface of the ER (2–5). It is not known how Golgi stacks manage to keep their structural integrity while moving through the cytoplasm, but the existence of a plant Golgi matrix has been proposed after electron microscopy revealed the existence of intercisternal elements and a ribosome-excluding zone around the stacks (6,7). Golgi matrix proteins could also be involved in tethering Golgi stacks to the ER surface (8).
The golgins, a Golgi-localized protein family with long coiled-coil domains, are ideal candidate proteins for being part of a putative Golgi matrix, as in animal cells golgins have been implied in formation and maintenance of Golgi stack structure (9–11). One example is the Golgi reassembly stacking proteins (GRASPs) that are involved in stacking of Golgi cisternae (12,13). Many golgins, such as the cis-Golgi localized p115 and GM130, have multiple tethering functions and can act in multiprotein tethering complexes with other golgins, small GTPases and SNARE proteins (reviewed in 14). Examples are the p115-giantin-GM130-Rab1 tether and the golgin-84-CASP (CCAAT-displacement protein alternatively spliced product) tether, which are found on coat protein I (COPI) membranes and could define different subpopulations of COPI vesicles (15). Homologues of golgins have been identified in Arabidopsis and localized to the Golgi apparatus, but their function remains largely unknown (16–22), although the Arabidopsis homologue of p115 has been suggested to play a role in tethering the Golgi stack to the ER (23).
Whereas some golgins are targeted to the Golgi stack via their C-terminal transmembrane domains, peripheral golgins obtain Golgi localization by interaction with other golgins or small regulatory GTPases. One example is the human peripheral coiled-coil protein TATA element modulatory factor (TMF) that binds to Rab6 (24) and was displaced from the Golgi stack upon RNAi-mediated depletion of the Rab protein (25). Other peripheral golgins contain a conserved C-terminal sequence consisting of ∼42 amino acids. This domain was originally identified in four mammalian golgins and named GRIP (golgin-97, RanBP2a, Imh1p, and p230/golgin-245) domain according to the starting letters of those proteins (26,27). The GRIP domain is highly conserved in eukaryotes and is required for targeting of proteins to the trans-Golgi or trans Golgi network (TGN) through interaction with the ARL1 (ARF-like) GTPases (28,29).
Although the organization of the plant Golgi apparatus differs significantly from that of yeast or animal cells (30), some basic structural mechanisms appear to be conserved, as several interactions between golgins and regulatory proteins have been identified in different species. For example, the interaction between human TMF and three isoforms of Rab6 (24) was also observed in yeast between the TMF homologue Sgm1p and the Rab6 homologue YPT6 (31) and in Arabidopsis between the homologues of Rab6, AtRab-H1b /c(32) and the homologue of TMF, GC5 (golgin candidate 5, 22). GRIP domain proteins appear to interact with the small GTPase ARL1 in yeast and animals (29,33,34) as well as in plants (19,21). AtGRIP shows high homology to the mammalian golgin-97, which contains a conserved C-terminal GRIP domain that interacts with Rab6 and is necessary and sufficient to target the protein to the trans-Golgi in mammalian cells (35).
Coiled-coil proteins have not only been implied in transport pathways between the ER and the Golgi but also in trafficking between the TGN and post-Golgi compartments. In yeast, the multisubunit tethering complex GARP (Golgi-associated retrograde protein) is involved in retrograde transport from endosomes to the TGN (36,37). This complex consists of the four subunits Vps51/52/53/54p, each containing coiled-coil domains (36), and has been shown to interact with Ypt6, the yeast homologue of mammalian Rab6 (38). Similarly, it has been shown that Vps52 interacts with Rab6 in mammalian cells (39). In Arabidopsis, a gene termed pok (poky pollen tube) identified in a mutant screen shares significant homology with the yeast Vps52p gene (40). POK/AtVPS52 appears to be part of a large protein complex and located to the Golgi, pre-vacuolar compartment and an unidentified post-Golgi compartment (41), but nothing is known to date about possible interactions between POK and the Arabidopsis homologues RabH1b and RabH1c.
Characterizing the interactions between golgins and regulatory or other structural proteins provides a first step towards a better understanding of their putative role within the Golgi stack. In vitro techniques such as yeast two-hybrid screens and pull down assays (reviewed in 42,43) can provide a general indication whether two proteins have the potential to interact with each other. Such techniques can, however, be compromised by the occurrence of false positive results and the artificial environment in which the binding takes place. Proteins interacting in vitro may never do so in vivo because they might be spatially separated in different cellular compartments (44). Therefore, it is preferable that in vitro results are confirmed using in vivo approaches.
A range of techniques are available to study protein–protein interactions in vivo using fluorescence microscopy. In the bimolecular fluorescence complementation (BiFC) assay, a fluorophore is split into two non-functional halves fused to two proteins of interest and interaction between the proteins restores the fluorescent signal (45,46). Analysis of FRET (Förster or fluorescence resonance energy transfer) between a donor and an acceptor fluorophore fused to two proteins of interest is more commonly used. FRET occurs under the conditions of a sufficiently large spectral overlap between donor emission and acceptor absorption spectrum, a favourable orientation of fluorophores to each other in a proximity of 1–10 nm (47). Interacting proteins are close enough to allow non-radiative energy transfer from the donor to the acceptor fluorophore, leading to quenching of donor fluorescence and an increase in acceptor fluorescence. Besides spectral bleed-through, other problems of this technique include the dependence on fluorophore expression levels and unintended photobleaching during measurements (48).
A method that combines FRET with fluorescence lifetime imaging (FLIM) has certain advantages over conventional FRET measurements (49–52). A fluorophore has a characteristic lifetime that remains unaffected by fluorophore concentration levels or excitation intensity, and spectral bleed-through does not constitute a problem as only the donor lifetime is measured (47). The fluorophore lifetime can be influenced by changes in temperature, environment, calcium ion concentration and the occurrence of FRET (51). Quenching of the donor fluorophore lifetime indicates interaction with the acceptor fluorophore and can be measured directly. FRET-FLIM has been successfully used in planta to study protein–protein interactions in protoplasts of cowpea (53–55), petunia (56), tobacco (57), maize (58) and Arabidopsis (59,60) as well as in barley (61) and tobacco leaf epidermal cells (62,63). Whereas FRET-FLIM has been used successfully on animal cells to visualize interactions between Rab GTPases and Rab binding proteins (64,65), in plants no such in vivo analyses of Rab or golgin protein interactions have been performed so far to our knowledge.
In this study we aimed to confirm in planta interactions between several plant Golgi tethering factors and small GTPases that to date only had been performed in vitro, by analysing the fluorescence lifetime (FLIM) after FRET by two-photon excitation (66). In this system, fluorophores are excited by the absorption of multiphotons in the near-infrared wavelength, which allows deeper penetration of tissues and leads to less cell damage. Absorption is confined to a narrow focussed plane and lacks out-of-focus excitation, therefore resulting in higher resolution and further reduction of photo damage (67).