The Lactose Permease q/'Escherichia coli: A Paradigm for Membrane 
Transport Proteins 
401 in the lacY gene. With respect to transport, 
permease molecules truncated at residue 396 or 
397 are completely defective, while those trun- 
cated at 398 through 401 exhibit 15-25, 30-40, 
40-45, and 70-100 percent of wild-type activ- 
ity, respectively. Wild-type permease or per- 
mease truncated at position 401 is stable, while 
molecules truncated at residues 400 down to 396 
are degraded at increasingly rapid rates. Thus ei- 
ther the last turn of putative helix XII or the re- 
gion immediately distal is important for pro- 
per folding and protection against proteolytic 
degradation. 
When the lacY gene is restricted into two ap- 
proximately equal-size fragments and subcloned 
individually or together under separate lac opera- 
tor/promoters, the permease is expressed in two 
portions: 1) the amino terminus, the first six pu- 
tative transmembrane helices, and most of puta- 
tive loop 7; and 2) the last six putative transmem- 
brane helices and the carboxyl terminus. 
Remarkably, cells expressing both fragments 
transport lactose to a steady-state level of accu- 
mulation at about 30 percent of the rate of cells 
expressing intact permease. In contrast, cells ex- 
pressing either portion of the permease indepen- 
dently do not transport lactose. 
Since intact permease is completely absent 
from the membrane of cells expressing /acFfrag- 
ments either individually or together, transport 
activity must result from an association between 
independently synthesized pieces of lac per- 
mease. If the gene fragments are expressed indi- 
vidually, the amino-terminal portion of the 
permease is observed sporadically and the car- 
boxyl-terminal portion is not observed. When the 
gene fragments are expressed together, polypep- 
tides identified as the amino- and carboxyl-termi- 
nal moieties of the permease are found in the 
membrane. The results indicate that the amino- 
or carboxyl-terminal halves of lac permease are 
proteolyzed when synthesized independently 
and that association between the two comple- 
menting polypeptides leads to a more stable, cata- 
lytically active complex. More recent experi- 
ments demonstrate that the permease can be split 
in a similar fashion, with comparable results, be- 
tween putative helix I and the rest of the protein 
or within putative helix VII. 
Notwithstanding the importance of high- 
resolution structure, site-directed mutagenesis 
can be used to delineate amino acid residues im- 
portant for active transport. Over 100 single- 
amino acid replacements have been made in the 
permease, and about 70 percent of the mutations 
have no significant effect on permease activity. 
Therefore it is unlikely that individual amino acid 
replacements result in large conformational alter- 
ations. Arg302 (putative helix IX) and Lys319, 
His, and Glu325 (all putative helix X) are criti- 
cally involved in lactose-coupled transloca- 
tion and/or substrate binding and recognition. 
Moreover, molecular modeling studies suggest 
that the four residues may be sufficiently close to 
form an H-bond network. 
These findings — the specific transport proper- 
ties of the mutants, and construction of mutants 
in which the putative geometric relationship be- 
tween the residues is altered — have led to the 
suggestion that the four residues may function in 
a type of H^-relay mechanism. In contrast, Cys 
residues, long thought to play a central role, are 
not involved in substrate binding or transloca- 
tion. Thus the Cys residues in the permease can 
be mutagenized individually or simultaneously, 
and significant activity is retained. 
228 
