There are two foci for this lab: how hydrophilic molecules (amino acids, proteins, mRNA, DNA) are targeted within a cell, and the causes of drug resistance in tumor cells. A battery of techniques from different disciplines (biochemistry, biophysics, electrophysiology, fluorescence, genetics, microscopy, molecular biology) is used to address these problems.

Protein Targeting. Eukaryotic cells contain numerous organelles, each enveloped by its own membrane. A membrane must form an effective sea wall of sorts, separating organelle from cytosol. But, since no organelle is entirely self-sufficient (or, to strain the metaphor, an island), the membrane surrounding it must also be permeable, allowing the in- and egress of various ions, sugars, nucleotides and proteins. Every organelle, for instance, must import proteins, which are synthesized in the cytosol. It's a two-step process. First, the proteins must be targeted to their destination. Then, they must cross the membrane. Those proteins that will become integral to the membrane must, further, get stitched into the bilayer in their proper topography.

Ions are known to cross membranes through aqueous channels. Our work indicates that, similarly, proteins cross membranes through transmembrane aqueous channels (Simon and Blobel, 1991, 1992). The questions we are currently addressing include:

• Are protein-conducting channels used in all cases where proteins cross membranes?
• How are transmembrane proteins integrated into the bilayer?
• Are transmembrane aqueous channels used for importing sugars, nucleotides, or for the uptake of DNA during transfection?

Protein transport is a fundamental cellular process which is essential for secretion, intercellular communication and organelle biogenesis, and, our observations have numerous clinical implications. Our studies on the biogenesis of opsin have characterized the etiology of some forms of progressive blindness and one of the transporters we are studying moves peptides for antigen presentation.

Multidrug Resistance in Tumors. A major failure of chemotherapy is the development of multidrug resistance in tumors. We have been studying some of the cell biological changes that occur in tumor cells that have developed resistance to chemotherapy. Using biochemistry, cell biology (cell fractionation and novel microscopic techniques), and biophysics (studies of membrane transport), we have characterized a number of distinct changes that characterize the drug-sensitive and drug-resistant states. Our results suggest novel strategies for chemotherapy which are proving successful in reversing drug resistance in in vitro assays.