@article{DondapatiLuebberdingZemellaetal.2019,
  author    = {Dondapati, Srujan Kumar and L{\"u}bberding, Henning and Zemella, Anne and Thoring, Lena and W{\"u}stenhagen, Doreen Anja and Kubick, Stefan},
  title     = {Functional Reconstitution of Membrane Proteins Derived From Eukaryotic Cell-Free Systems},
  series = {Frontiers in pharmacology},
  volume    = {10},
  journal   = {Frontiers in pharmacology},
  publisher = {Frontiers Research Foundation},
  address   = {Lausanne},
  issn      = {1663-9812},
  doi       = {10.3389/fphar.2019.00917},
  pages     = {9},
  year      = {2019},
  abstract  = {Cell-free protein synthesis (CFPS) based on eukaryotic Sf21 lysate is gaining interest among researchers due to its ability to handle the synthesis of complex human membrane proteins (MPs). Additionally Sf21 cell-free systems contain endogenous microsomal vesicles originally derived from the endoplasmic reticulum (ER). After CFPS, MPs will be translocated into the microsomal vesicles membranes present in the lysates. Thus microsomal membranes offer a natural environment for de novo synthesized MPs. Despite the advantage of synthesizing complex MPs with post translational modifications directly into the microsomal membranes without any additional solubilization supplements, batch based Sf21 cell-free synthesis suffers from low yields. The bottleneck for MPs in particular after the synthesis and incorporation into the microsomal membranes is to analyze their functionality. Apart from low yields of the synthesized MPs with batch based cell-free synthesis, the challenges arise in the form of cytoskeleton elements and peripheral endogenous proteins surrounding the microsomes which may impede the functional analysis of the synthesized proteins. So careful sample processing after the synthesis is particularly important for developing the appropriate functional assays. Here we demonstrate how MPs (native and batch synthesized) from ER derived microsomes can be processed for functional analysis by electrophysiology and radioactive uptake assay methods. Treatment of the microsomal membranes either with a sucrose washing step in the case of human serotonin transporter (hSERT) and sarco/endoplasmic reticulum Ca2+/ATPase (SERCA) pump or with mild detergents followed by the preparation of proteoliposomes in the case of the human voltage dependent anionic channel (hVDAC1) helps to analyze the functional properties of MPs.},
  language  = {en}
}
@article{RocchettiSharmaWulfetangeetal.2012,
  author    = {Rocchetti, Alessandra and Sharma, Tripti and Wulfetange, Camilla and Scholz-Starke, Joachim and Grippa, Alexandra and Carpaneto, Armando and Dreyer, Ingo and Vitale, Alessandro and Czempinski, Katrin and Pedrazzini, Emanuela},
  title     = {The putative K+ channel subunit AtKCO3 forms stable dimers in arabidopsis},
  series = {Frontiers in plant science},
  volume    = {3},
  journal   = {Frontiers in plant science},
  publisher = {Frontiers Research Foundation},
  address   = {Lausanne},
  issn      = {1664-462X},
  doi       = {10.3389/fpls.2012.00251},
  pages     = {13},
  year      = {2012},
  abstract  = {The permeation pore of K+ channels is formed by four copies of the pore domain. AtKCO3 is the only putative voltage-independent K+ channel subunit of Arabidopsis thaliana with a single pore domain. KCO3-like proteins recently emerged in evolution and, to date, have been found only in the genus Arabidopsis (A. thaliana and A. lyrata). We show that the absence of KCO3 does not cause marked changes in growth under various conditions. Only under osmotic stress we observed reduced root growth of the kco3-1 null-allele line. This phenotype was complemented by expressing a KCO3 mutant with an inactive pore, indicating that the function of KCO3 under osmotic stress does not depend on its direct ability to transport ions. Constitutively overexpressed AtKCO3 or AtKCO3::G FP are efficiently sorted to the tonoplast indicating that the protein is approved by the endoplasmic reticulum quality control. However, vacuoles isolated from transgenic plants do not have significant alterations in current density. Consistently, both AtKCO3 and AtKCO3::GFP are detected as homodimers upon velocity gradient centrifugation, an assembly state that would not allow for activity. We conclude that if AtKCO3 ever functions as a K+ channel, active tetramers are held by particularly weak interactions, are formed only in unknown specific conditions and may require partner proteins.},
  language  = {en}
}