ABSTRACT
Recent efforts to identify plasma membrane transporters mediating the selective uptake
of specific classes of organic anions have employed expression cloning techniques.
The transporters identified by these procedures have, almost without exception, differed
from putative transporters identified earlier by classical methods. In this review,
the classical and molecular approaches to the identification of membrane transporters
are examined and compared, with particular attention paid to the results obtained
by each with respect to sulfobromophthalein, bilirubin, and fatty acid transport.
The classical approach requires the initial identification and purification of a candidate
transport protein, proof of its function as a transporter by antibody inhibition or
liposome reconstitution studies, and, following the cloning of its cDNA, genetic expression
in transfected mammalian cells or in Xenopus laevis oocytes. Expression cloning affords
more rapid identification of proteins which (by definition) influence uptake, avoids
several potential artifacts (e.g., of conventional cDNA library screening techniques),
and yields rapid access to sequence information and derived structural characteristics.
However, it is ultimately necessary to express and purify the re-combinant protein
product, produce antibodies against synthetic peptides and/or the purified recombinant
protein, use the antibodies to identify the subcellular location of the cloned protein,
demonstrate that the protein binds the ligand of interest, and document that the protein
mediates a facilitated process with the characteristics of the one under study. Hence,
the two approaches ultimately require similar efforts. It is argued that the different
putative BSP/bilirubin and fatty acid transporters identified by the two approaches
may mediate different parallel transport pathways.
KEY WORDS
carrier-mediated transport - cloning - expression - transfection
