Molecular Basis and Functional Consequences of the Dominant Effects of the Mutant Band 3 on the Structure of Normal Band 3 in Southeast Asian Ovalocytosis

؊ exchange by an AE1 missense mutant devoid of CA2 binding, with activity further enhanced by CA2 co-expression. The same transport-incompetent AE1 mutants failed to rescue Cl ؊ /HCO 3 ؊ exchange by the AE1 truncation mutant 896X, despite preservation of the latter's core CA2 binding site. These data increase the minimal extent of a functionally defined CA2 binding site in AE1. The inter-protomeric rescue of HCO 3 ؊ transport within the AE1 dimer shows functional proximity of the C-terminal cytoplasmic tail of one protomer to the anion translocation pathway in the adjacent protomer within the AE1 heterodimer. The data strongly support the hypothesis that an intact transbilayer anion translocation pathway is completely contained within an AE1 monomer.

Section: Functional Effects Of Sequential Truncation Of the Kae1 C-supporting

confidence: 90%

Section: Interprotomeric Rescue Of Ae1-mediated Hcosupporting

confidence: 87%

Section: Interprotomeric Rescue Of Ae1-mediated Hcomentioning

confidence: 99%

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Deficient HCO3- Transport in an AE1 Mutant with Normal Cl- Transport Can be Rescued by Carbonic Anhydrase II Presented on an Adjacent AE1 Protomer

Dahl¹,

Jiang

Chernova

et al. 2003

“…The (43) as accessible to aqueous medium. However, the accessibility of the Pro 815 -Lys 829 region to extracellular medium is controversial.…”

Section: Discussionmentioning

confidence: 99%

Novel Topology in C-terminal Region of the Human Plasma Membrane Anion Exchanger, AE1

Zhu

Lee

Casey

2003

139

178

Human AE1 performs electroneutral exchange of Cl ؊ for HCO 3 ؊ across the erythrocyte membrane. We examined the topology of the AE1 C-terminal region using cysteine-scanning mutagenesis and sulfhydryl-specific chemistry. Eighty individual cysteine residues, introduced into an otherwise cysteine-less mutant between were inaccessible to the extracellular medium and thus localized to the intracellular surface of AE1. Functional assays revealed that one face of each of two AE1 TMs was sensitive to mutation. Based on these results, we propose a topology model for the C-terminal region of the membrane domain of human AE1.

“…It is not known whether these differences in the final membrane structure are due to differences in the topogenic functions of M1, the P region, and M2 in the two proteins. In ion transport proteins, the amino acid sequence forming the ion-conducting pathway is often surrounded by other segments of the polypeptide (18,19). However, information on how the pore is formed and becomes embedded in the membrane is limited.…”

mentioning

confidence: 99%

Topogenesis of Two Transmembrane Type K+ Channels, Kir 2.1 and KcsA

Umigai

Sato

Mizutani

et al. 2003

Potassium channels, which control the passage of K ؉ across cell membranes, have two transmembrane segments, M1 and M2, separated by a hydrophobic P region containing a highly conserved signature sequence. Here we analyzed the membrane topogenesis characteristics of the M1, M2, and P regions in two animal and bacterial two-transmembrane segment-type K ؉ channels, Kir 2.1 and KcsA, using an in vitro translation and translocation system. In contrast to the equivalent transmembrane segment, S5, in the voltage-dependent K ؉ channel, KAT1, the M1 segment in KcsA, was found to have a strong type II signal-anchor function, which favors the N cyt /C exo topology. The N-terminal cytoplasmic region was required for efficient, correctly orientated integration of M1 in Kir 2.1. Analysis of N-terminal modification by in vitro metabolic labeling showed that the N terminus in Kir 2.1 was acetylated. The hydrophobic P region showed no topogenic function, allowing it to form a loop, but not a transmembrane structure in the membrane; this region was transiently exposed in the endoplasmic reticulum lumen during the membrane integration process. M2 was found to possess a stop-transfer function and a type I signalanchor function, enabling it to span the membrane. The C-terminal cytoplasmic region in KcsA was found to affect the efficiency with which the M2 achieved their final structure. Comparative topogenesis studies of Kir 2.1 and KcsA allowed quantification of the relative contributions of each segment and the cytoplasmic regions to the membrane topology of these two proteins. The membrane topogenesis of the pore-forming structure is discussed using results for Kir 2.1, KcsA, and KAT1.