Multiple Roles of ADP-Ribosylation Factor 1 in Plant Cells Include Spatially Regulated Recruitment of Coatomer and Elements of the Golgi Matrix

Abstract

Recent evidence indicates that ADP-ribosylation factor 1 (ARF1) carries out multiple roles in plant cells that may be independent from the established effector complex COPI. To investigate potential COPI-independent functions, we have followed the dynamics of ARF1 and a novel putative effector, the plant golgin GRIP-related ARF-binding domain-containing Arabidopsis (Arabidopsis thaliana) protein 1 (GDAP1) in living plant cells. We present data that ascribe a new role to ARF1 in plant cell membrane traffic by showing that the GTPase functions to recruit GDAP1 to membranes. In addition, although ARF1 appears to be central to the recruitment of both COPI components and the golgin, we have established a different subcellular distribution of these ARF1 effectors. Live cell imaging demonstrates that GDAP1 and COPI are distributed on Golgi membranes. However, GDAP1 is also found on ARF1-labeled structures that lack coatomer, suggesting that the membrane environment, rather than ARF1 alone, influences the differential recruitment of ARF1 effectors. In support of this hypothesis, fluorescence recovery after photobleaching analyses demonstrated that GDAP1 and COPI have different kinetics on membranes during the cycle of activation and inactivation of ARF1. Therefore, our data support a model where modulation of the cellular functions of ARF1 in plant cells encompasses not only the intrinsic activities of the effectors, but also differential recruitment onto membranes that is spatially regulated.

Advances in Fluorescent Protein-Based Imaging for the Analysis of Plant Endomembranes

“…ARF1 is known to recruit coatomer to Golgi membranes. However, it has also been shown recently that ARF1 is capable of recruiting an additional effector, the golgin GDAP1, to the Golgi and post-Golgi organelles in plant cells (Matheson et al, 2007). FRAP analyses on fluorescent protein fusions of ARF1, coatomer, and GDAP1 have shown that the cycles of binding and release of ARF1 to and from the Golgi apparatus are similar to those of GDAP1 but faster than coatomer (Fig.…”

“…In studies of plant endomembranes, FRAP has been used relatively recently in establishing the nature of the factors that influence protein traffic dynamics from the ER to the Golgi apparatus (Brandizzi et al, 2002b) and the dynamics of the proteins that regulate ER-to-Golgi protein transport (daSilva et al, 2004;Yang et al, 2005). With a similar approach, it has been recently shown that the small GTPase ARF1 binds to and is released from Golgi membranes at a rapid turnover rate (Stefano et al, 2006;Matheson et al, 2007). ARF1 is known to recruit coatomer to Golgi membranes.…”

“…The generation of an intact YFP molecule can be considered an irreversible process (Magliery et al, 2005); a stable interaction may be established between the two proteins fused to the YFP halves. Although this may not be a problem for proteins that dissociate with a very slow turnover, the formation of stably interacting protein couples may lead to physiological artifacts for those protein-protein events that are based on fast dissociation rates, for example, the interaction of a GTPase-like ARF1 with its effectors (Stefano et al, 2006;Matheson et al, 2007). Therefore, during the course of an experiment (i.e.…”

“…1). With a similar approach, Matheson et al (2007) used triple labeling based on GFP5, yellow fluorescent protein (YFP), and the styryl-dye FM4-64 to determine the localization of the small GTPase ADP-ribosylation factor 1 (ARF1) with respect to putative endocytic compartments and the Golgi apparatus.…”

Syt1p promotes activation of Arl1p at the late Golgi to recruit Imh1p

“…RGA was then calculated as a percentage of the total fluorescence of both areas: RGS(PS/C)ϫ100. Measurements were performed on images acquired with the same microscope intensity settings (Matheson et al, 2007).…”