Exo70 Proteins Research Articles

Glucose-induced ERM protein activation and translocation regulates insulin secretion

A key step in regulating insulin secretion is insulin granule trafficking to the plasma membrane. Using live-cell time-lapse confocal microscopy, we observed a dynamic association of insulin granules with filamentous actin and PIP2-enriched structures. We found that the scaffolding protein family ERM, comprising ezrin, radixin, and moesin, are expressed in β-cells and target both F-actin and PIP2. Furthermore, ERM proteins are activated via phosphorylation in a glucose- and calcium-dependent manner. This activation leads to a translocation of the ERM proteins to sites on the cell periphery enriched in insulin granules, the exocyst complex docking protein Exo70, and lipid rafts. ERM scaffolding proteins also participate in insulin granule trafficking and docking to the plasma membrane. Overexpression of a truncated dominant-negative ezrin construct that lacks the ERM F-actin binding domain leads to a reduction in insulin granules near the plasma membrane and impaired secretion. Conversely, overexpression of a constitutively active ezrin results in more granules near the cell periphery and an enhancement of insulin secretion. Diabetic mouse islets contain less active ERM, suggestive of a novel mechanism whereby impairment of insulin granule trafficking to the membrane through a complex containing F-actin, PIP2, Exo70, and ERM proteins contributes to defective insulin secretion.

Recycling endosomes

Recycling endosomes

Crystal Structure of the S. cerevisiae Exocyst Component Exo70p

The exocyst is an evolutionarily conserved multiprotein complex required for the targeting and docking of post-Golgi vesicles to the plasma membrane. Through its interactions with a variety of proteins, including small GTPases, the exocyst is thought to integrate signals from the cell and signal that vesicles arriving at the plasma membrane are ready for fusion. Here we describe the three-dimensional crystal structure of one of the components of the exocyst, Exo70p, from Saccharomyces cerevisiae at 3.5A resolution. Exo70p binds the small GTPase Rho3p in a GTP-dependent manner with an equilibrium dissociation constant of approximately 70 microM. Exo70p is an extended rod approximately 155 angstroms in length composed principally of alpha helices, and is a novel fold. The structure provides a first view of the Exo70 protein family and provides a framework to study the molecular function of this exocyst component.

Interaction of BIG2, a brefeldin A-inhibited guanine nucleotide-exchange protein, with exocyst protein Exo70

Guanine nucleotide-exchange proteins activate ADP-ribosylation factors by accelerating the replacement of bound GDP with GTP. Mammalian brefeldin A-inhibited guanine nucleotide-exchange proteins, BIG1 and BIG2, are important activators of ADP-ribosylation factors for vesicular trafficking. To identify proteins that interact with BIG2, we used cDNA constructs encoding BIG2 sequences in a yeast two-hybrid screen of a human heart library. Clone p2-5-3, encoding a form of human exocyst protein Exo70, interacted with BIG2 amino acids 1-643 and 1-832, but not 644-832, which was confirmed by coimmunoprecipitation of in vitro-translated BIG2 N-terminal segments and 2-5-3. By immunofluorescence microscopy, endogenous BIG2 and Exo70 in HepG2 cells were visualized at Golgi membranes and apparently at the microtubule-organizing center (MTOC). Both were identified in purified centrosomes. Immunoreactive Exo70 and BIG2 partially or completely overlapped with gamma-tubulin at the MTOC in cells inspected by confocal microscopy. In cells incubated with brefeldin A, most of the BIG2, Exo70, and trans-Golgi protein p230 were widely dispersed from their perinuclear concentrations, but small amounts always remained, apparently at the MTOC. After disruption of microtubules with nocodazole, BIG2 and Exo70 were widely distributed in cells and remained only partially colocalized with p230, BIG2 more so than Exo70. We conclude that in HepG2 cells BIG2 and Exo70 interact in trans-Golgi network and centrosomes, as well as in exocyst structures or complexes that move along microtubules to the plasma membrane, consistent with a functional association in both early and late stages of vesicular trafficking.

Rho3 of Saccharomyces cerevisiae, which regulates the actin cytoskeleton and exocytosis, is a GTPase which interacts with Myo2 and Exo70.

The Rho3 protein plays a critical role in the budding yeast Saccharomyces cerevisiae by directing proper cell growth. Rho3 appears to influence cell growth by regulating polarized secretion and the actin cytoskeleton, since rho3 mutants exhibit large rounded cells with an aberrant actin cytoskeleton. To gain insights into how Rho3 influences these events, we have carried out a yeast two-hybrid screen using an S. cerevisiae cDNA library to identify proteins interacting with Rho3. Two proteins, Exo70 and Myo2, were identified in this screen. Interactions with these two proteins are greatly reduced or abolished when mutations are introduced into the Rho3 effector domain. In addition, a type of mutation known to produce dominant negative mutants of Rho proteins abolished the interaction with both of these proteins. In contrast, Rho3 did not interact with protein kinase C (Pkc1), an effector of another Rho family protein, Rho1, nor did Rho1 interact with Exo70 or Myo2. Rho3 did interact with Bni1, another effector of Rho1, but less efficiently than with Rho1. The interaction between Rho3 and Exo70 and between Rho3 and Myo2 was also demonstrated with purified proteins. The interaction between Exo70 and Rho3 in vitro was dependent on the presence of GTP, since Rho3 complexed with guanosine 5'-O-(3-thiotriphosphate) interacted more efficiently with Exo70 than Rho3 complexed with guanosine 5'-O-(3-thiodiphosphate). Overlapping subcellular localization of the Rho3 and Exo70 proteins was demonstrated by indirect immunofluorescence. In addition, patterns of localization of both Exo70 and Rho3 were altered when a dominant active allele of RHO3, RHO3(E129,A131), which causes a morphological abnormality, was expressed. These results provide a direct molecular basis for the action of Rho3 on exocytosis and the actin cytoskeleton.