ARF1 localizes to the golgi and the trans-golgi network.

The recruitment of coat proteins for transport vesicles (COPI-, COPII-, and clathrincoated) is mediated by the small GTPases of the ADP-ribosylation factor (ARF) family of which there are three SAR, 12 ARF, and six ARL genes in the Arabidopsis thaliana genome (Vernoud et al., 2003). These GTPases are themselves recruited by guanidine exchange factors located at the donor compartments, which convert them into active GTP-bound forms (Anders and Jürgens, 2008). The well-established inhibitor of ARF guanidine exchange factors is the fungal toxin brefeldin A (Robinson et al., 2008). Normally, the ARF-GTPases are located on membranes where transport vesicles are formed (Kawasaki et al., 2005; D’Souza-Schorey and Chavrier, 2006). According to the wealth of data from various eukaryotic organisms, these are the endoplasmic reticulum (ER) for COPII vesicles, Golgi apparatus for COPI vesicles, and the trans-Golgi network (TGN)/early endosome (EE) and the plasma membrane for clathrincoated vesicles. However, a recent article in The Plant Cell (Böhlenius et al., 2010) reported that a barley (Hordeum vulgare) green fluorescent protein (GFP)-tagged ARF (Hv-ARFA1b/1c) localizes to a multivesicular body (MVB) when transiently expressed in onion epidermal cells. We take issue with this conclusion, in particular because no electron microscopy evidence was presented to confirm that the structure labeled was indeed multivesiculate. Here, we present evidence that Arabidopsis ARF1 localizes to the TGN and Golgi stack. We argue that multiple lines of evidence are required to make an accurate determination of protein localization, especially with respect to the endomembrane system, which has high potential for overexpression and tagging-induced artifacts (Millar et al., 2009; Moore and Murphy, 2009). In all other higher plants so far investigated, principally Arabidopsis and tobacco (Nicotiana tabacum), the very similar AtARFA1c (differing from the barley ARF by only four amino acids) locates to the Golgi apparatus/TGN. A Golgi localization was first demonstrated using an antibody generated against a full-length At-ARFA1cglutathione S-transferase fusion protein (Pimpl et al., 2000) by both immunogold electron microscopy (Pimpl et al., 2000; Stierhof and El Kasmi, 2010) (Figures 1A and 1B) and immunofluorescence (Ritzenthaler et al., 2002) (Figures 1D, 1F, and 1G). The same result was obtained when ARF1(X)FP constructs are expressed (Xu and Scheres, 2005; Stefano et al., 2006; Densow et al., 2010) (Figures 1D and 1E). Functional confirmation of an ARF1 localization at the Golgi apparatus comes additionally from several expression studies on tobacco cells with GDP-(T31N) or GTP-(Q71L) restricted ARF1 mutants, which lead to an abrogation of ER-to-Golgi transport and a redistribution of Golgi enzymes into the ER (Lee et al., 2002; Takeuchi et al., 2002; Stefano et al., 2006). The expression of these mutants (leading to an inhibition of COPI vesicle formation) is equivalent to treatment with brefeldin A, which in tobacco results in a fusion of Golgi membranes with the ER (Langhans et al., 2011). However, there is good evidence that ARF1 also locates to the TGN. This was originally suggested by Pimpl et al. (2003) who showed that a functional ARF1 was also required for post-Golgi sorting of soluble vacuolar proteins, implying (as in mammals) that ARF1 was required for clathrin-coated vesicle formation at the TGN. Visualization of a potential ARF1 localization at the TGN was provided later by immunofluorescence in Arabidopsis using lines expressing SYP61–cyan fluorescent protein and VHA-a1-GFP as TGN markers (Paciorek et al., 2005; Tanaka et al., 2009) and demonstration that a FM4-64 positive compartment, distinct from the Golgi apparatus, was labeled by ARF1-GFP (Xu and Scheres, 2005; Tanaka et al., 2009). A similar, non-Golgi ARF1positive compartment was also identified by Stefano et al. (2006) and later confirmed as the TGN by immunogold electron microscopy by Stierhof and El-Kasmi (2010) (Figure 1A). Böhlenius et al. (2010) based their conclusion that Hv-ARF1 localizes to MVBs on the observation of ARF1 colocalization with the Arabidopsis Rab GTPase ARA7/RabF2 in a transient expression assay in onion epidermal cells. We cannot confirm this observation with At-ARF1 in Arabidopsis roots. At-ARF1 antibodies that specifically colocalize with stably expressed ARF1-GFP (Figure 1 D) did not label structures which carry stably expressed ARA7-GFP (Figure 1G). On the other hand, Xu and Scheres (2005) localized a small proportion of the ARF1-GFP signal in Arabidopsis root cells to an ARA7-positive organelle. This may reflect localization of a portion of ARA7 to the TGN/ EE since immunogold electron microscopy of plastic sections cut from high-pressure frozen Arabidopsis roots has confirmed the localization of ARA7 to the boundary membrane ofMVBs (Haas et al., 2007). ARA7 also has been detected at the TGN inArabidopsis roots (Stierhof and El Kasmi, 2010) and in a FM4-64-positive compartment, distinct from the Golgi apparatus (Dhonukshe et al., 2006). Taken together, all of the localization data so far available using different markers as well immunogold electron microscopy indicate that the reported colocalizations of ARF1 and ARA7 reflect the presence of both of these regulators at the TGN/EE rather than at the MVB. Is there experimental support for the speculation made by Böhlenius et al. (2010) that ARF1 at the MVB “mediates formation of vesicles with another destination, potentially in retrograde trafficking?” More precisely stated: Is there any evidence for (1) COPIor clathrin-coated vesicle formation at the MVB and (2) recycling from MVB/late endosomes (LEs)? In the mammalian literature, there are numerous papers implicating the role of various coatomer subunits in endocytosis (Whitney et al., 1995; Razi et al., 2009). However, COPI seems mainly to be involved in early trafficking events (e.g., effecting transferrin internalization and recycling from EE; Daro www.plantcell.org/cgi/doi/10.1105/tpc.110.082099 This article is a Plant Cell Advance Online Publication. The date of its first appearance online is the official date of publication. The article has been edited and the authors have corrected proofs, but minor changes could be made before the final version is published. Posting this version online reduces the time to publication by several weeks.