Cyclodextrins (CyDs) at higher concentrations were found to cause hemolysis of human erythrocytes in the order of beta- greater than alpha- greater than gamma-CyD in isotonic solution. Biphasic effects of CyDs were observed for the osmotic and heat-induced hemolysis; i.e. the protection at relatively low CyD concentrations and stimulation at higher CyD concentrations. From the scanning electron microscopic observations, CyDs induced shape changes of membrane internalization type on erythrocytes. CyDs caused the release of cholesterol from erythrocyte membrane in the order of beta- greater than gamma- greater than alpha-CyD. These results clearly indicate that CyD-induced hemolysis is probably a secondary event resulting from the membrane disruption which elicited the removal of membrane components from erythrocytes.

Rice (Oryza sativa) glutelins are synthesized on the endoplasmic reticulum as a precursor, which are then transported via the Golgi to protein storage vacuoles (PSVs), where they are proteolytically processed into acidic and basic subunits. The glutelin precursor mutant6 (glup6) accumulates abnormally large amounts of proglutelin. Map-base cloning studies showed that glup6 was a loss-offunction mutant of guanine nucleotide exchange factor (GEF), which activates Rab GTPase, a key regulator of membrane trafficking. Immunofluorescence studies showed that the transport of proglutelins and a-globulins to PSV was disrupted in glup6 endosperm. Secreted granules of glutelin and a-globulin were readily observed in young glup6 endosperm, followed by the formation of large dilated paramural bodies (PMBs) containing both proteins as the endosperm matures. The PMBs also contained membrane biomarkers for the Golgi and prevacuolar compartment as well as the cell wall component, b-glucan. Direct evidence was gathered showing that GLUP6/GEF activated in vitro GLUP4/Rab5 as well as several Arabidopsis (Arabidopsis thaliana) Rab5 isoforms to the GTP-bound form. Therefore, loss-of-function mutations in GEF or Rab5 disrupt the normal transport of proglutelin from the Golgi to PSVs, resulting in the initial extracellular secretion of these proteins followed, in turn, by the formation of PMBs. Overall, our results indicate that GLUP6/GEF is the activator of Rab5 GTPase and that the cycling of GTP-and GDP-bound forms of this regulatory protein is essential for the intracellular transport of proglutelin and a-globulin from the Golgi to PSVs and in the maintenance of the general structural organization of the endomembrane system in rice seeds.

Many GTPases regulate intracellular transport and signaling in eukaryotes. Guanine nucleotide exchange factors (GEFs) activate GTPases by catalyzing the exchange of their GDP for GTP. Here we present crystallographic and biochemical studies of a GEF reaction with four crystal structures of Arabidopsis thaliana ARA7, a plant homolog of Rab5 GTPase, in complex with its GEF, VPS9a, in the nucleotide-free and GDP-bound forms, as well as a complex with aminophosphonic acid-guanylate ester and ARA7⅐VPS9a(D185N) with GDP. Upon complex formation with ARA7, VPS9 wedges into the interswitch region of ARA7, inhibiting the coordination of Mg 2؉ and decreasing the stability of GDP binding. The aspartate finger of VPS9a recognizes GDP ␤-phosphate directly and pulls the P-loop lysine of ARA7 away from GDP ␤-phosphate toward switch II to further destabilize GDP for its release during the transition from the GDP-bound to nucleotide-free intermediates in the nucleotide exchange reaction.Small GTPases work as a molecular switch, which is turned off by its intrinsic GTPase activity hydrolyzing GTP to GDP. To turn the switch on, the bound GDP should be removed to introduce a GTP. Normally, this exchange reaction is much slower than the rate of intrinsic GTPase activity because nucleotide binds to small GTPase tightly with the Mg 2ϩ ion. GEF 5 enhances the nucleotide exchange of its cognate GTPase by destabilizing the Mg 2ϩ ion and GDP binding to the GTPase. Recognition of the GDP-bound GTPase by GEF is a critical but transient step prior to the formation of a nucleotide-free GTPase⅐GEF binary complex (1, 2). Because the nucleotide-free complex is stable in vitro, most GTPase⅐GEF complexes have been crystallized and structurally analyzed as the nucleotidefree form in the past, with a few exceptions containing nucleotides. One of them is a structure of eFF1A⅐eEF1B␣ in complex with different di-and triphosphate nucleotides, in which ␤-and ␥-phosphate electron densities of the nucleotides were ambiguous and only GMP could have been modeled (3). Another example is the Arf1⅐Sec7 complex, in which introducing either an abortive inhibitor or a mutation on the GEF allowed for GDP binding into the Arf1⅐Sec7 complex (4, 5). The complex structure between ROP4⅐GDP, a member of the plant Rho family, and its GEF PRONE8 indicated that GDP bound to the ROP4⅐PRONE8 complex loosely (6). A complex between Cdc42 and the DHR2 GEF domain of DOCK9 GEF has been crystallized in three different nucleotide forms: nucleotide-free, GDPbound, and GTP/Mg 2ϩ -bound. These structures demonstrate that the nucleotide sensor found in the ␣10 helix of the DHR2 domain is responsible for the exclusion of GDP/Mg 2ϩ and the introduction of GTP/Mg 2ϩ (7). It should be noted that none of the nucleotides were directly recognized by the GEFs in the complex structures mentioned above. These exceptions have encouraged us to investigate the transient nucleotide recognition in the GEF-catalyzed reaction.Rab small GTPases regulate vesicular transport in eukaryotes (8, 9), including ...