Abstract. Signal recognition particle (SRP) and SRP receptor are known to be essential components of the cellular machinery that targets nascent secretory proteins to the endoplasmic reticulum (ER) membrane. Here we report that the SRP receptor contains, in addition to the previously identified and sequenced 69-kD polypeptide (Qt-subunit, SRet), a 30-kD 13-subunit (SRJ]).When SRP receptor was purified by SRP-Sepharose affinity chromatography, we observed the co-purification of two other ER membrane proteins. Both proteins are ~30 kD in size and are immunologically distinct from each other, as well as from SRa and SRP proteins. One of the 30-kD proteins (SR~) forms a tight complex with SR~t in detergent solution that is stable to high salt and can be immunoprecipitated with antibodies to either SRct or SR~. Both subunits are present in the ER membrane in equimolar amounts and co-fractionate in constant stoichiometry when rough and smooth liver microsomes are separated on sucrose gradients. We therefore conclude that SRI3 is an integral component of SRP receptor. The presence of SRI~ was previously masked by proteolytic breakdown products of SRct observed by others and by the presence of another 30-kD ER membrane protein (mp30) which co-purifies with SRct. Mp30 binds to SRP-Sepharose directly and is present in the ER membrane in several-fold molar excess of SRa and SRI3. The affinity of mp30 for SRP suggests that it may serve a yet unknown function in protein translocation.

To select against glutamic acid or glutamate, both hydrogen atoms of the nitrogen of the glutamine sidechain are recognized. The hydroxyl group of Tyr211 and a water molecule are responsible for this recognition; both are obligate hydrogen-bond acceptors due to a network of interacting sidechains and water molecules. The prior binding of tRNAGln that is required for amino acid activation may result from the terminal nucleotide, A76, packing against and orienting Tyr211, which forms part of the amino acid binding site.

Sorbitol dehydrogenase (hSDH) and aldose reductase form the polyol pathway that interconverts glucose and fructose. Redox changes from overproduction of the coenzyme NADH by SDH may play a role in diabetes-induced dysfunction in sensitive tissues, making SDH a therapeutic target for diabetic complications. We have purified and determined the crystal structures of human SDH alone, SDH with NAD(+), and SDH with NADH and an inhibitor that is competitive with fructose. hSDH is a tetramer of identical, catalytically active subunits. In the apo and NAD(+) complex, the catalytic zinc is coordinated by His69, Cys44, Glu70, and a water molecule. The inhibitor coordinates the zinc through an oxygen and a nitrogen atom with the concomitant dissociation of Glu70. The inhibitor forms hydrophobic interactions to NADH and likely sterically occludes substrate binding. The structure of the inhibitor complex provides a framework for developing more potent inhibitors of hSDH.

We have identified the binding site of a new class of allosteric HLGP inhibitors. The crystal structure revealed the details of inhibitor binding, led to the design of a new class of compounds, and should accelerate efforts to develop therapeutically relevant molecules for the treatment of diabetes.