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Molecular genetics of
cation transporters. Prokaryotic and eukaryotic
cells contain a family of plasma-membrane ATPases that couple the energy from
ATP hydrolysis to the transport of ions across the membrane. These ATPases are
integral membrane proteins, approximately 100 kDa in size, with a cytoplasmic
nucleotide-binding domain and a membrane-embedded channel through which the transported
ions move. During the past few years, gene cloning and characterization have established
that the cytoplasmic domain has been relatively well conserved in evolution, while
the channel region has diverged to meet the physiological needs of particular
cells and tissues. We have adopted the PMA1 ATPase
of yeast as a simple model to study the biogenesis, function, and regulation of
this important group of transport proteins. Through the use of sec mutants with
temperature-sensitive blocks in the biogenesis pathway, we have found that maturation
of the ATPase is accompanied by the stepwise phosphorylation of multiple Ser and
Thr residues as the protein moves from its site of synthesis in the rough endoplastic
reticulum toward the cell surface. By the time the ATPase reaches the secretory
vesicles, it is fully competent to split ATP and pump protons; thus, the vesicles
provide an excellent way to express and characterize mutationally altered forms
of the protein, free of background contamination by pre-existing wild-type protein.
By means of this approach, we are carrying out site-directed mutagenesis to identify
amino acid residues that play an important role in the reaction cycle. 
Figure
caption: Topological model of the H+-ATPase in
the membrane, based on hydropathy analysis of its amino acid sequence. Solid
circles represent amino acid residues that are conserved among all known cation-transporting
ATPases; open circles represent residues that are similar (e.g., arginine and
lysine). Recent publications: Mason,
A.B., and Slayman, C.W. (2004) Plasma-membrane H+ pumps. In: Encyclopedia of Biological
Chemistry (G. Rice, ed.; Elsevier), in press.
Lecchi, S., and Slayman, C.W. (2004) Yeast plasma-membrane H+-ATPase: a model
system for studies of structure, function, biogenesis, and regulation. In: Handbook
of ATPases (M. Futai and J.H.Kaplan, eds.; Wiley-VCH), in press. Slayman,
C.W., Miranda, M. Pardo, J.P., and Allen, K.E. (2003) Use
of a fluorescent maleimide to probe structure-function relationships in stalk
segments 4 and 5 of the yeast plasma-membrane H+--ATPase. Ann. N.Y. Acad.
Sci. 986:168174.
Miranda,
M. Pardo, J.P., Allen, K.E., and Slayman, C.W. (2002) Stalk
segment 5 of the yeast plasma membrane H+--ATPase: labeling with a fluorescent
maleimide reveals a conformational change during glucose activation. J. Biol.
Chem. 277:40981-40988. Ferreira,
T., Mason, A.B., Pypaert, M., Allen, K.E., and Slayman, C.W. (2002) Quality
control in the yeast secretory pathway: a misfolded Pma1 H+-ATPase reveals two
checkpoints. J. Biol. Chem. 277:21027-21040. Ferreira
T, Mason AB, Slayman CW. The
yeast pma1 proton pump: a model for understanding the biogenesis of plasma membrane
proteins. J Biol Chem. 2001 Aug 10;276(32):29613-6.
Miranda M, Allen
KE, Pardo JP, Slayman CW. Stalk
segment 5 of the yeast plasma membrane H+-ATPase: mutational evidence for a role
in glucose regulation. J Biol Chem. 2001 Jun 22;276(25):22485-90.
Morsomme,
P., Slayman, C.W., and Goffeau (2000) Mutagenic
study of the structure, function, and biogenesis of the yeast plasma membrane
H+-ATPase. BBA Biomembrane Reviews, 2000 Nov 10; 1469:133-157.
carolyn.slayman@yale.edu
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