meric receptors found on a wide variety of mamma- 
lian cells. Analysis of this process has involved 
studying mutations located both in invasin and its 
integrin receptors that affect binding or uptake of 
bacteria. 
Dr. Guy Tran Van Nhieu in the laboratory has ana- 
lyzed the relationship between the binding of a li- 
gand to an integrin and bacterial uptake into host 
cells. He has shown that high-affinity binding is a 
critical determinant for bacterial uptake into mam- 
malian cells and that if there is insufficient affinity, 
the bacteria will simply adhere to the integrin with- 
out being internalized by the mammalian cell. To 
analyze the role of the integrin receptor in uptake, 
he has been analyzing mutations located in the (Sj- 
integrin chain, some of which were provided by 
Dr. Alan F. Horwitz (University of Illinois). There 
are two surprising results from these experiments. 
First, although uptake of the bacterium requires re- 
arrangement of the host cell cytoskeleton, the inte- 
grin is probably not binding the cytoskeleton during 
the uptake process. Second, factors involved in re- 
ceptor-mediated endocytosis are probably impor- 
tant for phagocytosis of microorganisms, and these 
factors must interact with the integrin during the 
bacterial uptake process. Studies are in progress to 
identify these factors that interact with the integrin 
receptor. The work on uptake of Y. pseudotubercu- 
losis was supported by a grant from the National 
Institute of Allergy and Infectious Diseases, National 
Institutes of Health. 
Dr. Isberg is also Associate Professor of Microbi- 
ology and Molecular Genetics at Tufts University 
School of Medicine, Boston. 
Articles 
Leong, J.M., Fournier, R.S., and Isberg, R.R. 1991. 
Mapping and topographic localization of epitopes 
of the Yersinia pseudotuberculosis invasin pro- 
tein. Infect Immun 59:3424-3433- 
Leong, J.M., Moitoso de Vargas, L.M., and Isberg, 
R.R. 1992. Binding of mammalian cells to immo- 
bilized bacteria. Infect Immun 60:683-686. 
Rankin, S., Isberg, R.R., and Leong, J. M. 1992. The 
integrin-binding domain of invasin is sufficient to 
allow bacterial entry into mammalian cells. Infect 
Immun 60:3909-3912. 
Tran Van Nhieu, G., and Isberg, R.R. 1991. The 
Yersinia pseudotuberculosis invasin protein and 
human fibronectin bind to mutually exclusive 
sites on the a^fi^ integrin receptor. / Biol Chem 
266:24367-24375. 
FUNCTIONAL INTERACTIONS BETWEEN INTRAMEMBRANE CHARGED RESIDUES 
IN THE LACTOSE PERMEASE OF ESCHERICHIA COLI 
H. Ronald Kaback, M.D., Investigator 
The lactose (lac) permease of Escherichia coli is 
a hydrophobic, polytopic cytoplasmic membrane 
protein that catalyzes the coupled translocation of 
18-galactosides and H^ with a 1:1 stoichiometry (i.e., 
symport or cotransport) . As such, this protein is a 
paradigm for a large number of secondary transport- 
ers that catalyze cation-coupled translocation reac- 
tions in diverse membranes ranging from those of 
archaebacteria to the mammalian central nervous 
system. Encoded by the lac Y gene, the permease has 
been solubilized from the membrane, purified, re- 
constituted into proteoliposomes, and shown to be 
solely responsible for iS-galactoside transpon, proba- 
bly as a monomer. Based on circular dichroic studies 
and hydropathy analysis of the deduced amino acid 
sequence, a secondary structure was proposed in 
which the permease has a short hydrophilic amino 
terminus, 1 2 hydrophobic domains in a-helical con- 
figuration that traverse the membrane in zigzag fash- 
ion connected by hydrophilic loops, and a 17- 
residue hydrophilic carboxyl-terminal tail. Evi- 
dence supporting the general features of the model 
and demonstrating that both the amino and carboxyl 
termini are on the cytoplasmic face of the mem- 
brane has been obtained from other spectroscopic 
techniques, limited proteolysis, immunological 
studies, and chemical modification. Analysis of an 
extensive series of lac permease-alkaline phospha- 
tase (JacY-phoA) fusions has provided exclusive 
support for the 12-helix motif. 
Recently it was found that lac permease mutants 
with Thr in place of Lys358 or Asn in place of 
Asp237 are defective with respect to active trans- 
port. Second-site suppressor mutations of K358T 
yield neutral amino acid substitutions for Asp237 
(Asn, Gly, or Tyr), while suppressors of D237N ex- 
hibit Gin in place of Lys358. Based on these find- 
ings, it was proposed that Asp237 and Lys358 inter- 
CELL BIOLOGY AND REGULATION 75 
