Over-expression of a mammalian small conductance calcium-activated K+ channel in Pichia pastoris: effects of trafficking signals and subunit fusions.
Mammalian SK proteins are Ca2+-activated K+ channels, which show a sub-20 pS conductance. We have expressed the SK2 variant gene in Pichia pastoris and found protein to be produced at considerably higher levels than in brain tissue. The channel was correctly folded as evidenced by its high affinity interaction with apamin, a specific ligand from bee venom. However, the protein was largely unable to reach the plasma membrane, its normal destination, instead remaining in the endoplasmic reticulum. Adding a putative translocation sequence altered the intracellular distribution significantly with enhanced trafficking out of the endoplamic reticulum. Fusion of SK2 with the associated protein calmodulin also altered the channel localisation but in a different manner with channels now found mainly in transit between endoplasmic reticulum and Golgi compartments.
Licata
Luana
L
Department of Structural Biology, Max Planck Institute for Biophysics, Frankfurt am Main, Germany.
Haase
Winfried
W
Eckhardt-Strelau
Luise
L
Parcej
David N
DN
D000818:Animals; D003001:Cloning, Molecular; D004721:Endoplasmic Reticulum / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008565:Membrane Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010843:Pichia / Q000235:genetics; D017510:Protein Folding; D021122:Protein Subunits / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D021381:Protein Transport / Q000235:genetics; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D015398:Signal Transduction / Q000235:genetics*; D051640:Small-Conductance Calcium-Activated Potassium Channels / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
D008565:Membrane Proteins; D021122:Protein Subunits; D011993:Recombinant Fusion Proteins; D051640:Small-Conductance Calcium-Activated Potassium Channels