Structure and Function of Vps15 in the Endosomal G Protein Signaling Pathway ,

G protein-coupled receptors mediate cellular responses to a wide variety of stimuli, including taste, light and neurotransmitters. In the yeast Saccharomyces cerevisiae, activation of the pheromone pathway triggers events leading to mating. The view had long been held that the G protein-mediated signal occurs principally at the plasma membrane. Recently, it has been shown that the G protein α subunit Gpa1 can promote signaling at endosomes and requires two components of the sole phosphatidylinositol-3-kinase in yeast, Vps15 and Vps34. Vps15 contains multiple WD repeats and also binds to Gpa1 preferentially in the GDP-bound state; these observations led us to hypothesize that Vps15 may function as a G protein β subunit at the endosome. Here we show an X-ray crystal structure of the Vps15 WD domain that reveals a seven-bladed propeller resembling that of typical Gβ subunits. We show further that the WD domain is sufficient to bind Gpa1 as well as to Atg14, a potential Gγ protein that exists in a complex with Vps15. The Vps15 kinase domain together with the intermediate domain (linking the kinase and WD domains) also contributes to Gpa1 binding, and is necessary for Vps15 to sustain G protein signaling. These findings reveal that the Vps15 Gβ-like domain serves as a scaffold to assemble Gpa1 and Atg14, whereas the kinase and intermediate domains are required for proper signaling at the endosome

Reversible folding of UDP-galactose 4-epimerase from Escherichia coli

UDP-galactose 4-epimerase from Escherichia coli is a homodimer of 39-kDa subunits having 1 or 2 molecules of NAD bound non-covalently/dimer. The enzyme can be dissociated and denatured by 8 M urea at pH 7.0 to a state having only 15% of residual secondary structure. Dilution of the denaturant by 20 mM potassium phosphate, pH 8.5, leads to functional reconstitution of the enzyme. No addition of extraneous NAD is required for reactivation, indicating a strong affinity of the cofactor for refolded molecule. The reactivation follows a second-order kinetics ( k = 1.2i0.07XlO' M-' s - ' at 25°C) with an energy of activation of 23.79 i 0.33 kJ/mol. The native, denatured and renatured states of the enzyme were characterized by far-ultraviolet CD spectra for secondary structure; protein fluorescence, interaction with extrinsic fluorescence probe ANS (1 -anilino 8-naphthalene sulfonic acid) and ultraviolet absorption spectra for tertiary structure and size-exclusion HPLC, gel-filtration chromatography and light-scattering for quaternary structure. The folding process could be broadly divided into two distinct steps: (a) regain of secondary structure and dimerization were fast and were complete within 2 min and 9 min, respectively, and (b) regain of catalytic activity was slow and was complete by 45 min. No active holoenzyme could be identified. It appears that generation of the NAD-binding site and subsequent assembly of NAD is the rate-limiting step expressing catalytic activity