Retromer is a peripheral membrane protein complex that has pleiotropic roles in endosomal membrane trafficking. The core of retromer possesses three subunits, VPS35, VPS29 and VPS26, that play different roles in binding to cargo, regulatory proteins and complex stabilization. We have performed an investigation of the thermodynamics of core retromer assembly using isothermal titration calorimetry (ITC) demonstrating that VPS35 acts as the central subunit to which VPS29 and VPS26 bind independently. Furthermore, we confirm that the conserved PRLYL motif of the large VPS35 subunit is critical for direct VPS26 interaction. Heat capacity measurements of VPS29 and VPS26 binding to VPS35 indicate extensive binding interfaces and suggest conformational alterations in VPS29 or VPS35 upon complex formation. Solution studies of the retromer core using small-angle X-ray scattering allow us to propose a model whereby VPS35 forms an extended platform with VPS29 and VPS26 bound at distal ends, with the potential for forming dimeric assemblies.

Cyclophilin 40 (CyP40) is a tetratricopeptide repeat (TPR)-containing immunophilin and a modulator of steroid receptor function through its binding to heat shock protein 90 (Hsp90). Critical to this binding are the carboxyl-terminal MEEVD motif of Hsp90 and the TPR domain of CyP40. Two different models of the CyP40-MEEVD peptide interaction were used as the basis for a comprehensive mutational analysis of the Hsp90-interacting domain of CyP40. Using a carboxyl-terminal CyP40 construct as template, 24 amino acids from the TPR and flanking acidic and basic domains were individually mutated by site-directed mutagenesis, and the mutants were coexpressed in yeast with a carboxyl-terminal Hsp90␤ construct and qualitatively assessed for binding using a ␤-galactosidase filter assay. For quantitative assessment, mutants were expressed as glutathione S-transferase fusion proteins and assayed for binding to carboxyl-terminal Hsp90␤ using conventional pulldown and enzyme-linked immunosorbent assay microtiter plate assays. Collectively, the models predict that the following TPR residues help define a binding groove for the MEEVD peptide: Lys-227, Asn-231, Phe-234, Ser-274, Asn-278, Lys-308, and Arg-312. Mutational analysis identified five of these residues (Lys-227, Asn-231, Asn-278, Lys-308, and Arg-312) as essential for Hsp90 binding. The other two residues (Phe-234 and Ser-274) and another three TPR domain residues not definitively associated with the binding groove (Leu-284, Lys-285, and Asp-329) are required for efficient Hsp90 binding. These data confirm the critical importance of the MEEVD binding groove in CyP40 for Hsp90 recognition and reveal that additional charged and hydrophobic residues within the CyP40 TPR domain are required for Hsp90 binding.In the absence of hormone, estrogen receptor ␣ is found predominantly in the nucleus as part of a multicomponent complex with Hsp90 1 (1). This abundant molecular chaperone plays an essential role in maintaining steroid receptors in a hormone-activable state, and the association of receptor with Hsp90 is a prerequisite for efficient signal transduction (1). In addition to dimeric Hsp90 and the receptor subunit, mature steroid receptor complexes contain p23 and one of a group of modulating proteins that include the immunophilins cyclophilin 40 (CyP40), FKBP51, and FKBP52 (1). CyP40 was first isolated as a component of estrogen receptor ␣ heterocomplexes (2). This novel cyclophilin binds the immunosuppressant cyclosporin A and shares structural similarity with its partner co-chaperones, FKBP51 and FKBP52, both immunophilins of the FK506-binding class (1). The structure of these large immunophilins is characterized by an amino-terminal immunophilin-like domain that is targeted by immunosuppressant drugs and overlaps a peptidylprolyl isomerase function and a carboxyl-terminal protein interaction domain incorporating 3 units of the tetratricopeptide repeat (TPR) motif (3). These immunophilins have been implicated in steroid receptor function through their association with Hsp...

SUMMARYStromules are highly dynamic stroma-filled tubules that extend from the surface of all plastid types in all multicellular plants examined to date. The stromule frequency (percentage of plastids with stromules) has generally been regarded as characteristic of the cell and tissue type. However, the present study shows that various stress treatments, including drought and salt stress, are able to induce stromule formation in the epidermal cells of tobacco hypocotyls and the root hairs of wheat seedlings. Application of abscisic acid (ABA) to tobacco and wheat seedlings induced stromule formation very effectively, and application of abamine, a specific inhibitor of ABA synthesis, prevented stromule induction by mannitol. Stromule induction by ABA was dependent on cytosolic protein synthesis, but not plastid protein synthesis. Stromules were more abundant in dark-grown seedlings than in light-grown seedlings, and the stromule frequency was increased by transfer of light-grown seedlings to the dark and decreased by illumination of dark-grown seedlings. Stromule formation was sensitive to red and far-red light, but not to blue light. Stromules were induced by treatment with ACC (1-aminocyclopropane-1-carboxylic acid), the first committed ethylene precursor, and by treatment with methyl jasmonate, but disappeared upon treatment of seedlings with salicylate. These observations indicate that abiotic, and most probably biotic, stresses are able to induce the formation of stromules in tobacco and wheat seedlings.