Abstract
BackgroundMembrane proteins define biological functions of membranes in cells. Extracellular peptides of transmembrane proteins receive signals from pathogens or environments, and are the major targets of drug developments. Despite of their essential roles, membrane proteins remain elusive in topological studies due to technique difficulties in their expressions and purifications.MethodsFirst, the target gene is cloned into a destination vector to fuse with C terminal ubiquitin at the N or C terminus. Then, Cub vector with target gene and NubWT or NubG vectors are transformed into AP4 or AP5 yeast cells, respectively. After mating, the diploid cells are dipped onto selection medium to check the growth. Topology of the target protein is determined according to Table 1.ResultsWe present a split ubiquitin topology (SUT) analysis system to study the topology and truncation peptide of membrane proteins in a simple yeast experiment. In the SUT system, transcription activator (TA) fused with a nucleo-cytoplasmic protein shows strong auto-activation with both positive and negative control vectors. TA fused with the cytoplasmic end of membrane proteins activates reporter genes only with positive control vector with a wild type N terminal ubiquitin (NubWT). However, TA fused with the extracellular termini of membrane proteins can’t activate reporter genes even with NubWT. Interestingly,TA fused with the released peptide of a membrane protein shows autoactivation in the SUT system.ConclusionThe SUT system is a simple and fast experimental procedure complementary to computational predictions and large scale proteomic techniques. The preliminary data from SUT are valuable for pathogen recognitions and new drug developments.
Highlights
Membrane proteins define biological functions of membranes in cells
A nucleo-cytoplasmic protein shows autoactivation in the split ubiquitin topology (SUT) system To validate the accuracy of the SUT system, a small soluble protein (GUS) is checked for auto-activation
In order to further clarify that the SUT system could distinguish membrane proteins, we applied this system to study four known transmembrane proteins, e.g. HPN and GPR3 (7 TM helices with extracellular N and cytosolic C termini) from human, VKOR (4 TM helices without cytosolic terminus) and PIP1;2 (6 TM helices, with two cytosolic termini) from Arabidopsis
Summary
Yeast split ubiquitin system The destination vector, pMetYC-Dest, and positive/ negative control vectors are available at Arabidopsis Research Center (www. arabidopsis.org). The target genes were cloned into pMetYN-Dest or pMetYC-Dest vectors by LR reactions using LR Clonase II as described in the user manual. The cell pellets were resuspended in 10 ml sterile water and centrifuged for another 5 min at 1000 g to remove the supernatant. WKS1 gene was cloned into pMDC43 and pMDC83 plasmids by LR reactions using LR Clonase II (Invitrogen) as described to construct N or C terminal GFP fusion proteins [25]. The cells were incubated at 30°C overnight in a shaker at 200 rpm, harvested by centrifugation at 1500 g for 5 min at RT and resuspended in Agrobacterium infiltration solution (10 mM MgCl2, 10 mM of MES and 200 mM acetosyringone) with OD600 around 0.8 for 3 h in RT.
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