TY - JOUR

T1 - Role of phylogenetically conserved amino acids in folding of Na,K-ATPase

AU - Jørgensen, J R

AU - Pedersen, P A

PY - 2001/6/19

Y1 - 2001/6/19

N2 - This paper focuses on the amino acid sequence 708-TGDGVNDSPALKK in pig kidney Na,K-ATPase as one of the best conserved among P-type ATPases. In Ca-ATPase this sequence forms a strand-loop-helix structure as part of a Rossman fold next to the phosphorylation site. Substitution of polar residues in the investigated sequence interfered with high-level accumulation of mutant protein. Mutant alpha1-subunit protein only accumulated in membranes from yeast cells grown at 15 degrees C whereas wild-type protein accumulated at both 15 and 35 degrees C. A systematic screen for the molecular mechanism behind lack of accumulation of mutant protein at 35 degrees C showed that transcription and translation were unaffected by the mutations. To demonstrate in vivo protein folding problems, an unfolded protein response reporter system was constructed in yeast. In this strain, only expression of mutant Na,K-ATPase alpha1-subunit caused induction of the unfolded protein response at 35 degrees C, indicating folding problems in the ER. Lowering the expression temperature to 15 degrees C prevented induction of the unfolded protein response after mutant protein expression, indicating correct folding at this temperature. At the permissive temperature mutant proteins were able to escape the endoplasmic reticulum quality control, reach the plasma membrane, and bind ouabain with high affinity. Since mutants in the 708-TGDGVNDSPALKK segment had a thermo inactivation profile identical to that of wild type, they were classified as temperature-sensitive synthesis mutants. The results indicate that this segment contributes side chains of importance for overall folding and maturation of Na,K-ATPase and all other P-type ATPases.

AB - This paper focuses on the amino acid sequence 708-TGDGVNDSPALKK in pig kidney Na,K-ATPase as one of the best conserved among P-type ATPases. In Ca-ATPase this sequence forms a strand-loop-helix structure as part of a Rossman fold next to the phosphorylation site. Substitution of polar residues in the investigated sequence interfered with high-level accumulation of mutant protein. Mutant alpha1-subunit protein only accumulated in membranes from yeast cells grown at 15 degrees C whereas wild-type protein accumulated at both 15 and 35 degrees C. A systematic screen for the molecular mechanism behind lack of accumulation of mutant protein at 35 degrees C showed that transcription and translation were unaffected by the mutations. To demonstrate in vivo protein folding problems, an unfolded protein response reporter system was constructed in yeast. In this strain, only expression of mutant Na,K-ATPase alpha1-subunit caused induction of the unfolded protein response at 35 degrees C, indicating folding problems in the ER. Lowering the expression temperature to 15 degrees C prevented induction of the unfolded protein response after mutant protein expression, indicating correct folding at this temperature. At the permissive temperature mutant proteins were able to escape the endoplasmic reticulum quality control, reach the plasma membrane, and bind ouabain with high affinity. Since mutants in the 708-TGDGVNDSPALKK segment had a thermo inactivation profile identical to that of wild type, they were classified as temperature-sensitive synthesis mutants. The results indicate that this segment contributes side chains of importance for overall folding and maturation of Na,K-ATPase and all other P-type ATPases.

KW - Alanine/genetics

KW - Amino Acid Substitution/genetics

KW - Amino Acids/genetics

KW - Animals

KW - Asparagine/genetics

KW - Cell Membrane/genetics

KW - Conserved Sequence

KW - Endoplasmic Reticulum/enzymology

KW - Enzyme Activation/genetics

KW - Gene Expression Regulation, Fungal

KW - Lysine/genetics

KW - Mutagenesis, Site-Directed

KW - Peptide Fragments/genetics

KW - Phylogeny

KW - Protein Folding

KW - RNA, Messenger/metabolism

KW - Recombinant Proteins/antagonists & inhibitors

KW - Saccharomyces cerevisiae/enzymology

KW - Serine/genetics

KW - Sodium-Potassium-Exchanging ATPase/antagonists & inhibitors

KW - Swine

KW - Temperature

KW - Threonine/genetics

M3 - Journal article

C2 - 11401578

VL - 40

SP - 7301

EP - 7308

JO - Biochemistry

JF - Biochemistry

SN - 0006-2960

IS - 24

ER -