Search

The glycosyltransferases of the mammalian Golgi complex must recycle between the stacked cisternae of that organelle to maintain their proper steady-state localization. This trafficking is mediated by COPI-coated vesicles, but how the glycosyltransferases are incorporated into these transport vesicles is poorly understood. Here we show that the N-terminal cytoplasmic tails (N-tails) of a number of cis Golgi glycosyltransferases which share a ϕ-(K/R)-X-L-X-(K/R) sequence bind directly to the δ- and ζ-subunits of COPI. Mutations of this N-tail motif impair binding to the COPI subunits, leading to mislocalization of the transferases to lysosomes. The physiological importance of these interactions is illustrated by mucolipidosis III patients with missense mutations in the N-tail of GlcNAc-1-phosphotransferase that cause the transferase to be rapidly degraded in lysosomes. These studies establish that direct binding of the N-tails of mammalian cis Golgi glycosyltransferases with COPI subunits is essential for recycling within the Golgi. [ABSTRACT FROM AUTHOR]

Two proteins, STIM1 in the endoplasmic reticulum and Orail in the plasma membrane, are required for the activation of Ca[sup2+] release-activated Ca[sup2+] (CRAC) channels at the cell surface. How these proteins interact to assemble functional CRAC channels has remained uncertain. Here, we determine how many Orail and STIM1 molecules are required to form a functional CRAC channel. We engineered several genetically expressed fluorescent Orail tandem multimers and a. fluorescent, constitutively active STIM1 mutant. The tandem multimers assembled into CRAC channels, as seen by rectifying inward currents and by cytoplasmic calcium elevations. CRAC channels were visualized as fluorescent puncta in total internal reflection microscopy. With single-molecule imaging techniques, it was possible to observe photo-bleaching of individual fluorophores and to count the steps of bleaching as a measure of the stoichiometry of each CRAC channel complex. We conclude that the subunit stoichiometry in an active CRAC channel is four Orail molecules and two STIM1 molecules. Fluorescence resonance energy transfer experiments also showed that four Orail subunits form the assembled channel. From the fluorescence intensity of single fluorophores, we could estimate that our transfected HEK293 cells had almost 400,000 CRAC channels and that, when intracellular Ca[sup2+] stores were depleted, the channels clustered in aggregates containing ≈1,3O0 channels, amplifying the local Ca[sup2+] entry. [ABSTRACT FROM AUTHOR]

Many multifunctional tumor suppressor proteins have low stability, a property linked to cancer development. The von Hippel-Lindau tumor suppressor protein (pVHL) is one of these proteins. pVHL forms part of the E3 ubiquitin ligase complex that regulates the degradation of the hypoxia-inducible factor (HIF). Under native conditions, free pVHL is a molten globule, but it is stabilized in the E3 complex. By using molecular dynamics simulations, we observed that the interface between the two pVHL domains is the least stable region in unbound pVHL. We designed five stable mutants: one with a mutation at the interdomain interface and the others in the α- or β-domains. Experimentally, type ZB pVHL disease mutant Y98N at the HIF binding site was shown to destabilize pVHL and decrease its binding affinity to HIF. Our simulations showed that the decrease in pVHL stability and binding affinity are allosterically regulated. The mutations designed to stabilize unbound wild-type pVHL which are away from the elongin C and HIF binding sites, successfully stabilized the Y98N pVHL-elongin C complex and lowered the binding free energy of pVHL with HIF. Our results indicated both the enthalpic and dynamic allosteric components between the elongin C and HIF binding sites in pVHL in the a- and p-domains, respectively, mediated by the interdomain interface and linker. Drugs mimicking the allosteric effects of these mutants may rescue pVHL function in von Hippel-Lindau disease. [ABSTRACT FROM AUTHOR]