The Lactose Permease q/*Escherichia coli: 
A Paradigm for Membrane Transport Proteins 
H. Ronald Kaback, M.D. — Investigator 
Dr. Kaback is also Professor of Physiology and Microbiology and Molecular Genetics in the Molecular Bi- 
ology 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 Bio- 
chemistry. Dr. Kaback is a member of the National Academy of Sciences. Among his honors is the Kenneth 
Cole Award of the American Biophysical Society. 
A critical basic biological problem that re- 
mains unsolved is the mechanism of energy 
transduction in biological membranes. A wide 
range of seemingly disparate phenomena, such as 
oxidative phosphorylation, bacterial motility, 
and solute accumulation against a concentration 
gradient (secondary active transport) , are driven 
by bulk-phase, transmembrane electrochemical 
H"*^ or Na"*" gradients. However, the molecular 
mechanism by which energy stored in such gra- 
dients is transduced into work or energy-rich 
compounds (e.g., ATP) remains unknown. In 
order to gain insight into this process, studies in 
our laboratory have focused on the lactose (lac) 
permease of Escherichia coli as a paradigm. 
The ability of E. coli to accumulate the disac- 
charide lactose and other |S-galactosides against a 
large concentration gradient depends on the lac 
permease, a very hydrophobic cytoplasmic mem- 
brane protein that catalyzes the coupled translo- 
cation of these sugars and with a stoichiome- 
try of unity (i.e., symport or co-transport). Under 
physiological conditions, where the electro- 
chemical gradient across the cytoplasmic mem- 
brane is interior negative and/or alkaline, lac 
permease utilizes free energy released from 
downhill translocation of to drive accumu- 
lated (8-galactosides against a concentration gra- 
dient, the magnitude of which is directly related 
to that of the H"^ gradient. In the absence of an 
gradient, the permease catalyzes the converse re- 
action, utilizing free energy released from down- 
hill translocation of /?-galactosides to drive uphill 
translocation of and generating an electro- 
chemical gradient, the polarity of which depends 
on the direction of the concentration gradient of 
the substrate. 
Encoded by the /acFgene — the second struc- 
tural gene in the lac operon — the permease has 
been solubilized from the membrane, purified to 
homogeneity, and reconstituted into proteolipo- 
somes in a fully functional state. In addition, we 
have presented evidence that the permease is 
functional as a monomer. Circular dichroic mea- 
surements demonstrating that purified permease 
is about 80 percent helical, and hydropathy analy- 
sis of the deduced amino acid sequence, suggest a 
secondary structure in which the protein is pre- 
dicted to have a short hydrophilic amino ter- 
minus, 12 hydrophobic domains in a-helical 
conformation that traverse the membrane in zig- 
zag fashion connected by hydrophilic loops, and 
a 17-residue hydrophilic carboxyl-terminal tail. 
Through other approaches, we have confirmed 
the general features of the model and demon- 
strated that the amino and carboxyl termini are on 
the cytoplasmic surface of the membrane. More- 
over, strong exclusive support for the topological 
predictions of the 1 2-helix model is provided by 
Calamia and Manoil's studies on an extensive se- 
ries of lac permease-alkaline phosphatase fusion 
proteins. 
The topology of polytopic membrane proteins 
is thought to result from either the orientation of 
the first amino-terminal hydrophobic domain in 
the membrane or from topogenic determinants 
dispersed throughout the molecules. We have 
now studied the insertion and stability of in- 
frame deletion mutants in lac permease. So long 
as the first and last putative a-helical domains are 
retained, stable polypeptides are inserted into 
the membrane, even when an odd number of he- 
lical domains are deleted. Moreover, when an 
odd number of helices are deleted, the carboxyl 
terminus remains on the membrane's cytoplas- 
mic surface. Thus relatively short carboxyl-termi- 
nal domains of the permease appear to contain 
topological information sufficient for insertion in 
the native orientation. Finally, permease mole- 
cules devoid of even or odd numbers of putative 
transmembrane helices retain a specific pathway 
for downhill lactose translocation. 
Previous experiments indicate that amino acid 
residues 396-40 1 at the carboxyl terminus of the 
last putative transmembrane helix of lac per- 
mease are important for protection against pro- 
teolytic degradation and suggest that this region 
of the permease may be necessary for proper fold- 
ing. Termination codons have now been substi- 
tuted sequentially for amino acid codons 396 to 
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