The conserved aspartic acid residues shown to be essential for enzymatic activity in yeast and mammalian lipins are indicated by asterisks (*). Subcellular localization of TbLpn To determine the subcellular this website localization of TbLpn, PF T. brucei cells were fractionated into cytosolic and nuclear extracts, and the presence of TbLpn within these compartments assessed by I-BET151 price Western hybridization. The efficiency of the fractionation procedure was confirmed by using antibodies directed against cytosolic Hsp70 and nuclear
RNA polymerase II. As shown in Figure 3, a band of the expected size for TbLpn (~ 83 kDa) was present exclusively in the cytoplasm of the parasite. This is in contrast to all previously characterized mammalian and yeast lipins which display cytoplasmic as well as nuclear localization [34, 39, 49–51]. In addition, SMP2, the yeast lipin homologue, has been shown to be present in the cytosol as
well as associated with the membrane . We did however detect the presence of a protein band with decreased electrophoretic mobility (~120 kDa) in the nuclear extract. This strongly suggests that TbLpn is present in both cytosol and nucleus and, in the nucleus, is heavily modified by post-translational modifications such as arginine methylation and/or phosphorylation. Figure 3 Analysis of TbLpn subcellular localization. PF T. brucei were fractionated into cytosolic VX-680 supplier (C) and nuclear (N) extracts as described under Material and Methods. The presence of TbLpn was detected by western hybridization using anti-TbLpn polyclonal antibodies (1:1,000), followed by goat anti-rabbit IgGs, and signals detected using chemiluminescence.
Efficiency of the fractionation procedure was assessed by western blot using antibodies against Hsp70 and RNA polymerase II as cytosolic and nuclear markers, respectively. TbLpn interacts with TbPRMT1 in vitro and in vivo We further confirmed the TbPRMT1/TbLpn interaction DCLK1 identified by yeast-two-hybrid first by Far Western hybridization. To this end, recombinant His-TbLpn was electrophoresed and transferred to PVDF, and the membrane was incubated with recombinant His-TbPRMT1. Detection of His-TbPRMT1 with polyclonal anti-TbPRMT1 antibodies revealed the presence of a band at 105 kDa, which is the predicted size of His-TbLpn, thereby demonstrating direct binding of His-TbPRMT1 to His-TbLpn (Figure 4A). As a negative control, His-RBP16, expressed and purified using the same protocol as for the purification of His-TbLpn, was used. Using this negative control, no band was detected. The data indicate that TbLpn and TbPRMT1 interact directly. Figure 4 TbLpn interacts with TbPRMT1. A) Far western analysis of TbPRMT1-TbLpn interaction. Purified His-TbLpn and His-RBP16 were separated on a 10% polyacrylamide gel, transferred to PVDF, and incubated with purified TbPRMT1 as described under Material and Methods.