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Use of a Fluorescent Maleimide to Probe Structure‐Function Relationships in Stalk Segments 4 and 5 of the Yeast Plasma‐Membrane H + ‐ATPase
Author(s) -
SLAYMAN CAROLYN W.,
MIRANDA MANUEL,
PARDO JUAN PABLO,
ALLEN KENNETH E.
Publication year - 2003
Publication title -
annals of the new york academy of sciences
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.712
H-Index - 248
eISSN - 1749-6632
pISSN - 0077-8923
DOI - 10.1111/j.1749-6632.2003.tb07156.x
Subject(s) - chemistry , membrane , atpase , yeast , stalk , biophysics , biochemistry , cytoplasm , maleimide , alexa fluor , fluorescence , enzyme , stereochemistry , biology , polymer chemistry , horticulture , physics , quantum mechanics
A bstract : In the yeast plasma‐membrane H + ‐ATPase and other P‐type ATPases, conformational changes are transmitted between cytoplasmic and membrane‐embedded domains via a stalk region composed of cytoplasmic extensions of membrane segments 2, 3, 4, and 5. The present study has used a fluorescent maleimide (Alexa‐488) to probe Cys residues introduced into stalk segments 4 and 5 of the yeast enzyme. In the case of S5, Cys substitutions along one face led to a constitutive, 5‐ to 10‐fold activation of the ATPase in the absence of glucose. Based on homology with SERCA Ca 2+ ‐ATPase, this face is likely to be buried in the interior of the protein, close to the P domain. Three Cys residues on the opposite face of S5 (A668C, S672C, and D676C) were accessible to Alexa‐488 under all conditions tested. In addition, three other Cys residues at or near the boundary between the two faces reacted with Alexa‐488 only (V665C, L678C) or preferentially (Y689C) in plasma membranes from glucose‐metabolizing cells; this result provides the first direct evidence for a change in conformation of S5 during glucose activation. For stalk segment 4, site‐directed mutagenesis gave no sign of a role in glucose‐dependent regulation. Rather, substitutions at 13 consecutive positions along S4 caused kinetic changes consistent with a shift in equilibrium from E2 to E1. Four Cys residues along this stretch of S4 (Q357C, K362C, S364C, and S368C) reacted with Alexa‐488, indicating that they are exposed to the aqueous medium as predicted in the SERCA‐based structural model.