Arabidopsis Profilin Isoforms, PRF1 and PRF2 Show Distinctive Binding Activities and Subcellular Distributions

Abstract

Profilin is an actin-binding protein that shows complex effects on the dynamics of the actin cytoskeleton. There are five profilin isoforms in Arabidopsis thaliana L. However, it is still an open question whether these isoforms are functionally different. In the present study, two profilin isoforms from Arabidopsis, PRF1 and PRF2 were fused with green fluorescent protein (GFP) tag and expressed in Escherichia coli and A. thaliana in order to compare their biochemical properties in vitro and their cellular distributions in vivo. Biochemical analysis revealed that fusion proteins of GFP-PRF1 and GFP-PRF2 can bind to poly-L-proline and G-actin showing remarkable differences. GFP-PRF1 has much higher affinities for both poly-L-proline and G-actin compared with GFP-PRF2. Observations of living cells in stable transgenic A. thaliana lines revealed that 35S::GFP-PRF1 formed a filamentous network, while 35S::GFP-PRF2 formed polygonal meshes. Results from the treatment with latrunculin A and a subsequent recovery experiment indicated that filamentous alignment of GFP-PRF1 was likely associated with actin filaments. However, GFP-PRF2 localized to polygonal meshes resembling the endoplasmic reticulum. Our results provide evidence that Arabidopsis profilin isoforms PRF1 and PRF2 have different biochemical affinities for poly-L-proline and G-actin, and show distinctive localizations in living cells. These data suggest that PRF1 and PRF2 are functionally different isoforms.

A Processive Arabidopsis Formin Modulates Actin Filament Dynamics in Association with Profilin

“…Expression plasmids for FH1-FH2, FH2 (Li et al, 2010), mDia1 FH1-FH2-C (Moseley et al, 2006), ABP29 (Xiang et al, 2007), AtPRF2 (Wang et al, 2009), AtPRF4 (Ye et al, 2009), LlPRF1 (Vidali and Hepler, 1997), and human profilin (Kovar et al, 2006) have been described previously. The SNAP coding region was PCR amplified from the SNAP-tag-T7-2-vector (NEB, Ipswitch, MA), and combined with FH1-FH2, FH2, ABP29, and mDia1-FH1-FH2-C by fusion PCR with a GGSGGS flexible linker between them.…”

Actin-Binding Proteins and Actin Dynamics in Plant Cells

“…Recently, PRF1 and PRF2, two Arabidopsis profilin isoforms, were fused with green fluorescent protein and stably expressed in Arabidopsis, and then their localization were observed in living cells of transgenic lines. The results show that PRF1-GFP is associated with a filamentous network in different cell types, but PRF2-GFP forms polygonal meshes closely resembling ER structures [161]. Nevertheless, the mechanism of these distinct distributions of pofilin isoforms in different plant cells and developmental stages has not been addressed yet.…”

“…To investigate the physiological function of profilins, the cellular localization of profilin in living cells from several different plant species has been studied by using immunocytochemical analyses [59,75,155], the microinjection of fluorescently labeled profilin [155] and the expressing the profilins fused with fluorescence protein in transgenic plants [161]. However, there are diverse localization of profilins in different cell types, profilin isoforms and analysis methods.…”

Profilin as a regulator of the membrane-actin cytoskeleton interface in plant cells

“…In Arabidopsis , observations of living cells in stable transgenic plants revealed that 35S:: GFP-AtPRF1 forms a filamentous network likely associated with actin filaments; this was verified by treatment with latrunculin A, and through a recovery experiment involving the removal of latrunculin A. Whereas, 35S:: GFP-AtPRF2 forms polygonal meshes resembling ER in the same latrunculin A treatment conditions (Wang et al, 2009). Furthermore, in plants, profilins possibly participate in the linkage of the nuclear envelope and the actin cytoskeleton during the interphase of Arabidopsis ; this is because AtFH8 locates primarily to the nuclear envelope at this stage (Xue et al, 2011).…”

“…The Arabidopsis profilin family includes five highly different isoforms: AtPRF1–AtPRF5; AtPRF1–AtPRF3 belong to the vegetative class, and AtPRF4 and AtPRF5 to the reproductive class (Christensen et al, 1996; Kandasamy et al, 2002). AtPRF1 has much higher affinities for both PLP and G-actin than AtPRF2 (Wang et al, 2009). The tobacco profilin gene, pronp1, is prominently expressed in mature pollen, elongating pollen tubes, and the root hairs of developing seedlings.…”