Energy-transducing Membrane Proteins 
H. Ronald Kaback, M.D. — Investigator 
Dr. Kaback is also Professor of Physiology and of Microbiology and Molecular Genetics in the Molecular 
Biology Institute of the University of California, Los Angeles. He received his M.D. degree from the Albert 
Einstein College of Medicine, interned at Bronx Municipal Hospital Center, and did postdoctoral research 
in physiology at Einstein. Subsequently he conducted research in membrane biochemistry at the National 
Heart Institute and the Roche Institute of Molecular Biology, chairing at Roche the Department of 
Biochemistry. Dr. Kaback is a member of the National Academy of Sciences and the American Academy 
of Arts and Sciences. Among his honors is the Distinguished Alumnus Award from the Albert Einstein 
College of Medicine. 
THE molecular mechanism of energy trans- 
duction in the membranes of living cells is an 
enigma. Although the immediate driving force 
for many seemingly unrelated processes, such as 
active transport, oxidative phosphorylation, and 
bacterial motility, is a bulk-phase, transmem- 
brane electrochemical H"^ or Na^ gradient, the 
molecular mechanism (s) by which free energy 
stored in these gradients is transduced into work 
or into other forms of chemical energy (e.g., 
ATP) remains unknown. In order to gain insight 
into this important basic problem, studies in our 
laboratory have focused on an enzyme, the lac- 
tose (lac) permease of the bacterium Escherichia 
coli, as a paradigm. 
The ability of E. coli to accumulate the sugar 
lactose and other |8-galactosides against a concen- 
tration gradient is dependent upon lac permease, 
a very hydrophobic plasma membrane protein 
that catalyzes the coupled translocation of a sin- 
gle sugar molecule with a single H"^ (i.e., symport 
or co-transport) . Under physiological conditions, 
lac permease utilizes free energy derived from 
downhill translocation of to drive accumu- 
lated (S-galactosides against a concentration gra- 
dient or, conversely, uses free energy released 
from downhill translocation of /J-galactosides to 
drive uphill translocation of H^. The polarity of 
the reaction reflects the direction of the concen- 
tration gradient of the substrate. As such, lac per- 
mease represents a huge number of machines that 
catalyze similar reactions in virtually all biologi- 
cal membranes, from archebacteria to the mam- 
malian central nervous system. 
The permease is encoded by the lacY gene, 
which has been cloned into a recombinant 
plasmid and sequenced. By combining overex- 
pression with the use of a specific photoaffinity- 
labeled substrate for the permease and recon- 
stitution of transport activity in artificial 
phospholipid vesicles (i.e., proteoliposomes), 
the permease was solubilized from the mem- 
brane, purified to homogeneity, and shown to cat- 
alyze all the transport reactions typical of the fi- 
galactoside transport system in vivo with similar 
turnover numbers. Therefore, a single enzyme — 
the product of lacY — is solely responsible for all 
of the translocation phenomena catalyzed by the 
iS-galactoside transport system. In addition, evi- 
dence has been presented that the permease is 
functional as a monomer. 
Based on circular dichroic measurements indi- 
cating that purified permease is about 80 percent 
helical and on hydropathy analysis of the de- 
duced amino acid sequence, a secondary struc- 
ture was proposed. The model predicts that the 
protein has a short hydrophilic amino terminus, 
12 hydrophobic domains in a-helical conforma- 
tion that traverse the membrane in zigzag fashion 
connected by hydrophilic loops, and a 17- 
residue hydrophilic carboxyl-terminal tail. Spec- 
troscopic, biochemical, and immunological data 
are consistent with the general features of the 
model and indicate that the amino and carboxyl 
termini are on the cytoplasmic surface of the 
membrane. Studies on an extensive series of lac 
permease-alkaline phosphatase chimeras have 
provided strong support for the topological pre- 
dictions of the 12-helix model. 
This report concentrates on current experi- 
ments that involve the use of site-directed muta- 
genesis to engineer lac permease so as to permit 
certain biochemical and biophysical approaches 
to structure-function relationships. 
Recent studies provide definitive support for 
the argument that cysteinyl residues do not play a 
direct role in the lac permease mechanism. Thus, 
when site-directed mutagenesis is used to replace 
each of the eight cysteinyl residues simulta- 
neously, the "C-Iess" permease catalyzes active 
lactose transport moderately well relative to 
wild-type permease (about 35 percent of the 
maximum velocity and 55 percent of the steady- 
state level of accumulation). Moreover, active 
lactose transport in right-side-out vesicles con- 
taining C-less permease is not inactivated by 
the alkylating agent, 7V-ethylmaleimide, in dra- 
matic contrast to vesicles containing wild-type 
permease. 
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