Isolation And Purification Of Profilin 2 Associated Proteins From Arabidopsis Endoplasmic Reticulum

The polymerization and depolymerization of actin filaments are highly regulated, spatially and temporally, so that cells are capable to remodel the cytoskeleton rapidly in response to endogenous cues or external signals. Profilin is one of the most important factors to regulate microfilament dynamics and organization in living cells. In Arabidopsis thaliana, there are five profilin isoforms expressed respectively in different plant organs and at different development stages. These isoforms may play unique functions in plant cells. Reported studies showed that profilin evenly distributes throughout the cytoplasm and does not specifically associate with any cellular structures. However, our previous study showed that profilin 1 and profilin 2 distributed distinguishably in cells. Analysis of two GFP-Profilin isoforms in transgenic Arabidopsis demonstrated that GFP:Profilin 1 exhibited as filamentous network, resembles the microfilaments, however, GFP:Profilin 2 presented a mesh network like endoplasmic reticulum (ER) in Arabidopsis cells. Such different distributions suggested that these two profilin isoforms may have different functions in cells. We presume that profilin 2 may associate with certain protein (s) located at the ER membrane.This study focuses on profilin 2, one of three profilins constitutively expressed in vegetative tissues, expecting to isolate some proteins specifically associated with profilin 2 from ER, and analysis these proteins by MS or animo acid sequencing. In present study, sucrose density gradient centrifugation was employed to purify the rough ER. The purification method was improved so that the higher activity of membrane proteins and the quantity of rER was obtained. Transmission electric microscopy indicated that the improved method was efficient and that the major component of separated membrane was rER. The binding experiment using the colloidal gold which coved by E. coli (DE3) expressed profilin 2 and purified rER demonstrated that profilin 2 may directly associate with ER by proteins at ER membrane. By pull-down experiment by purified recombinant profilin 1 and profilin 2, membrane proteins of rER specifically associate with profilin 2 were isolated. The analysis of SDS-PAGE and staining the gel by improved silver stain method identified a few bands of proteins which are specifically associated with profilin 2. One abundant protein with molecular mass of 28 kDa was chosen and analyzed by LC-MS/MS. The result showed that the unknown protein has fragment of polypeptide with the sequence of DFEEPGFLAPTGLFLGGEK, which matches profilin 1. Analysis suggested that this sequence in profilin 1 may has actin binding activity, so we presume that the 28 kDa unknown protein may have actin binding actibity, too. This study provides important evidence to show the relationship between profilin 2 and ER, and in further, to understand the regulatory function of profilin in ER dynamics.