Reconstitution of water channel function of an aquaporin overexpressed and purified from Pichia pastoris.
The aquaporin PM28A is one of the major integral proteins in spinach leaf plasma membranes. Phosphorylation/dephosphorylation of Ser274 at the C-terminus and of Ser115 in the first cytoplasmic loop has been shown to regulate the water channel activity of PM28A when expressed in Xenopus oocytes. To understand the mechanisms of the phosphorylation-mediated gating of the channel the structure of PM28A is required. In a first step we have used the methylotrophic yeast Pichia pastoris for expression of the pm28a gene. The expressed protein has a molecular mass of 32462 Da as determined by matrix-assisted laser desorption ionization-mass spectrometry, forms tetramers as revealed by electron microscopy and is functionally active when reconstituted in proteoliposomes. PM28A was efficiently solubilized from urea- and alkali-stripped Pichia membranes by octyl-beta-D-thioglucopyranoside resulting in a final yield of 25 mg of purified protein per liter of cell culture.
Karlsson
Maria
M
Department of Plant Biochemistry, Lund University, Box 124, Sweden. maria.karlsson@plantbio.lu.se
Fotiadis
Dimitrios
D
Sjövall
Sara
S
Johansson
Ingela
I
Hedfalk
Kristina
K
Engel
Andreas
A
Kjellbom
Per
P
D000818:Animals; D020346:Aquaporins / Q000235:genetics* / Q000502:physiology / Q000648:ultrastructure; D001483:Base Sequence; D017931:DNA Primers; D005260:Female; D007473:Ion Channels / Q000235:genetics* / Q000502:physiology / Q000648:ultrastructure; D007700:Kinetics; D009865:Oocytes / Q000502:physiology; D010766:Phosphorylation; D010768:Phosphoserine; D010843:Pichia / Q000235:genetics / Q000502:physiology*; D010940:Plant Proteins / Q000235:genetics / Q000502:physiology / Q000648:ultrastructure; D011510:Proteolipids / Q000378:metabolism; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism / Q000648:ultrastructure; D012694:Serine; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D018724:Spinacia oleracea; D014982:Xenopus laevis
D020346:Aquaporins; D017931:DNA Primers; D007473:Ion Channels; C100932:PM28A protein, Spinacia oleracea; D010940:Plant Proteins; D011510:Proteolipids; D011994:Recombinant Proteins; C020485:proteoliposomes; D010768:Phosphoserine; D012694:Serine