NaPi-IIa and interacting partners.

Regulation of renal proximal tubular reabsorption of phosphate (Pi) is one of the critical steps in Pi homeostasis. Experimental evidence suggests that this regulation is achieved mainly by controlling the apical expression of the Na+-dependent Pi cotransporter type IIa (NaPi-IIa) in proximal tubules. Only recently have we started to obtain information regarding the molecular mechanisms that control the apical expression of NaPi-IIa. The first critical observation was the finding that truncation of only its last three amino acid residues has a strong effect on apical expression. A second major finding was the observation that the last intracellular loop of NaPi-IIa contains sequence information that confers parathyroid hormone (PTH) sensitivity. The use of the above domains of the cotransporter in yeast two-hybrid (Y2H) screening allowed the identification of proteins interacting with NaPi-IIa. Biochemical and morphological, as well as functional, analyses have allowed us to obtain insights into the physiological roles of such interactions, although our present knowledge is still far from complete.

Interaction of the type IIa Na/Pi cotransporter with PDZ proteins.

The type IIa Na(+)-dependent inorganic phosphate (Na/P(i)) cotransporter is localized in the apical membrane of proximal tubular cells and is regulated by an endocytotic pathway. Because molecular processes such as apical sorting, internalization, or subsequent degradation might be assisted by associated proteins, a yeast two-hybrid screen against the C-terminal, cytosolic tail of type IIa cotransporter was designed. Most of the potential proteins found belonged to proteins with multiple PDZ modules and were either identical/related to PDZK1 or identical to NHERF-1. Yeast trap truncation assays confined the peptide-protein association to the C-terminal amino acid residues TRL of type IIa cotransporter and to single PDZ domains of each identified protein, respectively. The specificity of these interactions were confirmed in yeast by testing other apical localized transmembraneous proteins. Moreover, the type IIa protein was recovered in vitro by glutathione S-transferase-fused PDZ proteins from isolated renal brush border membranes or from type IIa-expressing oocytes. Further, these PDZ proteins are immunohistochemically detected either in the microvilli or in the subapical compartment of proximal tubular cells. Our results suggest that the type IIa Na/P(i) cotransporter interacts with various PDZ proteins that might be responsible for the apical sorting, parathyroid hormone controlled endocytosis or the lysosomal sorting of internalized type IIa cotransporter.

Sequence-specific rates of interaction of target peptides with the molecular chaperones DnaK and DnaJ.

The kinetics of complex formation between nine different fluorescence-labeled peptides (7-22 amino acid residues) and DnaK (Hsp70 homologue of Escherichia coli) in the nucleotide-free R state and in the ATP-liganded T state were measured. R-state DnaK (1 microM) formed high-affinity complexes (Kd = 0.06-2 microM) and bound all peptides (22-50 nM) in slow one- or two-step processes with apparent rate constants for the first phase, varying only by a maximum factor of 30 (kobs1 = 0.003-0.084 s-1 at pH 7.0 and 25 degreesC). In contrast, the rates of complex formation between DnaK-ATP and the same peptides (Kd = 2.2-107 microM) have been found previously to vary by 4 orders of magnitude [one- or two-step processes with kobs1 = 0.001-7.9 s-1; Gisler, S. M., Pierpaoli E. V., and Christen, P. (1998) J. Mol. Biol. 279, 833-840]. The slow and relatively uniform rates of peptide binding to the R state might be determined by the fraction of time during which the alpha-helical lid above the peptide-binding site is open. The faster and widely divergent rates of binding to the open T state might reflect sequence-specific conformational rearrangements in the peptide-binding site and perhaps of the peptide itself. The different rates of association with DnaK-ATP suggest a kinetic partitioning of target sequences in which only slowly interacting segments of polypeptides are channeled into the chaperone cycle.

Catapult mechanism renders the chaperone action of Hsp70 unidirectional.

Molecular chaperones of the Hsp70 type promote the folding and membrane translocation of proteins. The interaction of Hsp70s with polypeptides is linked to ATP binding and hydrolysis. We formed complexes of seven different fluorescence-labeled peptides with DnaK, the Hsp70 homolog of Escherichia coli, and determined the rate of peptide release under two different sets of conditions. (1) Upon addition of ATP to nucleotide-free peptide.DnaK complexes, all tested peptides were released with similar rate constants (2.2 s-1 to 6.7 s-1). (2) In the binding equilibrium of peptide and ATP-liganded DnaK, the dissociation followed one or two-step reactions, depending on the amino acid sequence of the peptide. For the monophasic reactions, the dissociation rate constants diverged by four orders of magnitude from 0.0004 s-1 to 5.7 s-1; for the biphasic reactions, the rate constants of the second, slower isomerization step were in the range from 0.3 s-1 to 0.0005 s-1. The release of the different peptides in case (1) is 1.4 to 14,000 times faster than in case (2). Apparently, binding of ATP induces a transient state of the chaperone which ejects target peptides before the final state of ATP-liganded DnaK is reached. This "catapult" mechanism provides the chaperone cycle with a mode of peptide release that does not correspond with the reverse of peptide binding. By allowing the conformation of the outgoing polypeptide to differ from that of the incoming polypeptide, a futile cycle with respect to conformational work exerted on the target protein is obviated.