Stefan Albert

Identification of the catalytic domains and their functionally critical arginine residues of two yeast GTPase-activating proteins specific for Ypt/Rab transport GTPases

Ypt/Rab proteins constitute the largest subfamily of the Ras superfamily of monomeric GTPases and are regulators of vesicular protein transport. Their slow intrinsic GTPase activity (10−4−10−3 min−1 at 30°C) has to be accelerated to switch the active to the inactive conformation. We have identified the catalytic domain within the C‐terminal halves of two yeast GTPase‐activating proteins (GAPs), Gyp1p and Gyp7p, with specificity for Ypt/Rab GTPases. The catalytically active fragments of Gyp1p and Gyp7p were more active than the full‐length proteins and accelerated the intrinsic GTP hydrolysis rates of their preferred substrates by factors of 4.5 × 104 and 7.8 × 105, respectively. The Km values for the Gyp1p and Gyp7p active fragments (143 and 42 μM, respectively) indicate that the affinities of those GAPs for their substrates are very low. The catalytic domains of Gyp1p and Gyp7p contain five invariant arginine residues; substitutions of only one of them (R343 in Gyp1p and R458 in the analogous position of Gyp7p) rendered the GAPs almost completely inactive. We suggest that Ypt/Rab‐GAPs, like Ras‐ and Rho‐GAPs, follow the same mode of action and provide a catalytic arginine (‘arginine finger’) in trans to accelerate the GTP hydrolysis rate of the transport GTPases.

Two new members of a family of Ypt/Rab GTPase activating proteins: promiscuity of substrate recognition.

Monomeric GTPases of the Ras superfamily have a very slow intrinsic GTPase activity which is accelerated by specific GTPase-activating proteins. In contrast to Ras- and Rho-specific GTPase-activating proteins (GAPs) that have been studied in great detail, little is known about the functioning of GAPs specific for Ypt/Rab transport GTPases. We have identified two novel Ypt/Rab-GAPs because of their sequence relatedness to the three known GAPs Gyp1p, Gyp6p, and Gyp7p. Mdr1/Gyp2p is an efficient GAP for Ypt6p and Sec4p, whereas Msb3/Gyp3p is a potent GAP for Sec4p, Ypt6p, Ypt51p, Ypt31/Ypt32p, and Ypt1p. Although the affinity of Msb3/Gyp3p for its preferred substrate Sec4p is low (K m = 154 μm), it accelerates the intrinsic GTPase activity of Sec4p 5 × 105-fold. Msb3/Gyp3p appears to be functionally linked to Cdc42p-regulated pathway(s). The results demonstrate that in yeast there is a large family of Ypt/Rab-GAPs, members of which discriminate poorly between GTPases involved in regulating different steps of exo- and endocytic transport routes.

Crystal structure of the GAP domain of Gyp1p: first insights into interaction with Ypt/Rab proteins.

We present the 1.9 Å resolution crystal structure of the catalytic domain of Gyp1p, a specific GTPase activating protein (GAP) for Ypt proteins, the yeast homologues of Rab proteins, which are involved in vesicular transport. Gyp1p is a member of a large family of eukaryotic proteins with shared sequence motifs. Previously, no structural information was available for any member of this class of proteins. The GAP domain of Gyp1p was found to be fully α‐helical. However, the observed fold does not superimpose with other α‐helical GAPs (e.g. Ras‐ and Cdc42/Rho‐GAP). The conserved and catalytically crucial arginine residue, identified by mutational analysis, is in a comparable position to the arginine finger in the Ras‐ and Cdc42‐GAPs, suggesting that Gyp1p utilizes an arginine finger in the GAP reaction, in analogy to Ras‐ and Cdc42‐GAPs. A model for the interaction between Gyp1p and the Ypt protein satisfying biochemical data is given.

Significance of GTP Hydrolysis in Ypt1p-regulated Endoplasmic Reticulum to Golgi Transport Revealed by the Analysis of Two Novel Ypt1-GAPs

We here report on the identification and detailed biochemical characterization of two novel GTPase-activating proteins, Gyp5p and Gyp8p, whose efficient substrate is Ypt1p, a Ypt/Rab-GTPase essential for endoplasmic reticulum-to-Golgi trafficking in yeast. Gyp5p accelerated the intrinsic GTPase activity of Ypt1p 4.2 × 104-fold and, surprisingly, the 40-fold reduced GTP hydrolysis rate of Ypt1(Q67L)p 1.5 × 104-fold. At steady state, the two newly discovered GTPase-activating proteins (GAPs) as well as the previously described Gyp1p, which also uses Ypt1p as the preferred substrate, display different subcellular localization. To add to an understanding of the significance of Ypt1p-bound GTP hydrolysis in vivo, yeast strains expressing the GTPase-deficient Ypt1(Q67L)p and having different Ypt1-GAP genes deleted were created. Depending on the genetic background, different mutants exhibited growth defects at low temperature and, already at permissive temperature, various morphological alterations resembling autophagy. Transport of proteins was not significantly impaired. Growth defects of Ypt1(Q67L)-expressing cells could be suppressed on high expression of all three Ypt1-GAPs. We propose that permanently active Ypt1p leads to increased vesicle fusion, which might induce previously unnoticed autophagic degradation of exaggerated membrane-enclosed structures. The data indicate that hydrolysis of Ypt1p-bound GTP is a prerequisite for a balanced vesicle flow between endoplasmic reticulum and Golgi compartments.

Immune-related proteins induced in the hemolymph after aseptic and septic injury differ in honey bee worker larvae and adults.

We have employed the proteomic approach in combination with mass spectrometry to study the immune response of honey bee workers at different developmental stages. Analysis of the hemolymph proteins of noninfected, mock-infected and immune-challenged individuals by polyacrylamide gel electrophoresis showed differences in the protein profiles. We present evidence that in vitro reared honey bee larvae respond with a prominent humoral reaction to aseptic and septic injury as documented by the transient synthesis of the three antimicrobial peptides (AMPs) hymenoptaecin, defensin1, and abaecin. In contrast, young adult worker bees react with a broader spectrum of immune reactions that include the activation of prophenoloxidase and humoral immune responses. At least seven proteins appeared consistently in the hemolymph of immune-challenged bees, three of which are identical to the AMPs induced also in larvae. The other four, i.e., phenoloxidase (PO), peptidoglycan recognition protein-S2, carboxylesterase (CE), and an Apis-specific protein not assigned to any function (HP30), are induced specifically in adult bees and, with the exception of PO, are not expressed after aseptic injury. Structural features of CE and HP30, such as classical leucine zipper motifs, together with their strong simultaneous induction upon challenge with bacteria suggest an important role of the two novel bee-specific immune proteins in response to microbial infections.

Reversible Membrane Interaction of BAD Requires two C-terminal Lipid Binding Domains in Conjunction with 14-3-3 Protein Binding

BAD is a Bcl-2 homology domain 3 (BH3)-only proapoptotic member of the Bcl-2 protein family that is regulated by phosphorylation in response to survival factors. Binding of BAD to mitochondria is thought to be exclusively mediated by its BH3 domain. We show here that BAD binds to lipids with high affinities, predominantly to negatively charged phospholipids, such as phosphatidylserine, phosphatidic acid, and cardiolipin, as well as to cholesterol-rich liposomes. Two lipid binding domains (LBD1 and LBD2) with different binding preferences were identified, both located in the C-terminal part of the BAD protein. BAD facilitates membrane translocation of Bcl-XL in a process that requires LBD2. Integrity of LBD1 and LBD2 is also required for proapoptotic activity in vivo. Phosphorylation of BAD does not affect membrane binding but renders BAD susceptible to membrane extraction by 14-3-3 proteins. BAD can be removed efficiently by 14-3-3ζ, -η, -τ and lesxs efficiently by other 14-3-3 isoforms. The assembled BAD·14-3-3 complex exhibited high affinity for cholesterol-rich liposomes but low affinity for mitochondrial membranes. We conclude that BAD is a membrane-associated protein that has the hallmarks of a receptor rather than a ligand. Lipid binding is essential for the proapoptotic function of BAD in vivo. The data support a model in which BAD shuttles in a phosphorylation-dependent manner between mitochondria and other membranes and where 14-3-3 is a key regulator of this relocation. The dynamic interaction of BAD with membranes is tied to activation and membrane translocation of Bcl-XL.

Regulation of RAF activity by 14-3-3 proteins: RAF kinases associate functionally with both homo- and heterodimeric forms of 14-3-3 proteins.

Mammalian 14-3-3 proteins play a crucial role in the activation process of RAF kinases. However, little is known about the selectivity of the mammalian 14-3-3 isoforms with respect to RAF association and activation. Using mass spectrometry, we analyzed the composition of the 14-3-3 isoforms attached to RAF kinases and found that B-RAF associates in vivo with 14-3-3 at much higher diversity than A- and C-RAF. We also examined in vitro binding of purified mammalian 14-3-3 proteins to RAF kinases using surface plasmon resonance techniques. While B- and C-RAF exhibited binding to all seven 14-3-3 isoforms, A-RAF bound with considerably lower affinities to ϵ, τ, and σ 14-3-3. These findings indicate that 14-3-3 proteins associate with RAF isoforms in a pronounced isoform-specific manner. Because 14-3-3 proteins appear in dimeric forms, we addressed the question of whether both homo- and heterodimeric forms of 14-3-3 proteins participate in RAF signaling. For that purpose, the budding yeast Saccharomyces cerevisiae, possessing only two 14-3-3 isoforms (BMH1 and BMH2), served as testing system. By deletion of the single BMH2 gene, we found that both homo- and heterodimeric forms of 14-3-3 can participate in RAF activation. Furthermore, we show that A-, B-, and C-RAF activity is differentially regulated by its C-terminal and internal 14-3-3 binding domain. Finally, prohibitin, a scaffold protein that affects C-RAF activation in a stimulatory manner, proved to interfere with the internal 14-3-3 binding site in C-RAF. Together, our results shed more light on the complex mechanism of RAF activation, particularly with respect to activation steps that are mediated by 14-3-3 proteins and prohibitin.

Identification of Novel in Vivo Phosphorylation Sites of the Human Proapoptotic Protein BAD PORE-FORMING ACTIVITY OF BAD IS REGULATED BY PHOSPHORYLATION

BAD is a proapoptotic member of the Bcl-2 protein family that is regulated by phosphorylation in response to survival factors. Although much attention has been devoted to the identification of phosphorylation sites in murine BAD, little data are available with respect to phosphorylation of human BAD protein. Using mass spectrometry, we identified here besides the established phosphorylation sites at serines 75, 99, and 118 several novel in vivo phosphorylation sites within human BAD (serines 25, 32/34, 97, and 124). Furthermore, we investigated the quantitative contribution of BAD targeting kinases in phosphorylating serine residues 75, 99, and 118. Our results indicate that RAF kinases represent, besides protein kinase A, PAK, and Akt/protein kinase B, in vivo BAD-phosphorylating kinases. RAF-induced phosphorylation of BAD was reduced to control levels using the RAF inhibitor BAY 43-9006. This phosphorylation was not prevented by MEK inhibitors. Consistently, expression of constitutively active RAF suppressed apoptosis induced by BAD and the inhibition of colony formation caused by BAD could be prevented by RAF. In addition, using the surface plasmon resonance technique, we analyzed the direct consequences of BAD phosphorylation by RAF with respect to association with 14-3-3 and Bcl-2/Bcl-XL proteins. Phosphorylation of BAD by active RAF promotes 14-3-3 protein association, in which the phosphoserine 99 represented the major binding site. Finally, we show here that BAD forms channels in planar bilayer membranes in vitro. This pore-forming capacity was dependent on phosphorylation status and interaction with 14-3-3 proteins. Collectively, our findings provide new insights into the regulation of BAD function by phosphorylation.

Age‐related and light‐induced plasticity in opsin gene expression and in primary and secondary visual centers of the nectar‐feeding ant Camponotus rufipes

Camponotus rufipes workers are characterized by an age‐related polyethism. In the initial weeks of adult life, young workers perform tasks inside the nest before they switch to multimodal foraging tasks outside. We tested the hypothesis that this transition is accompanied by profound adaptations in the peripheral and central visual systems. Our results show that C. rufipes workers of all tested ages (between 1 and 42 days) express three genes encoding for ultraviolet (UV), blue (BL), and long‐wavelength (LW1) sensitive opsins in their retina, which are likely to provide the substrate for trichromatic color vision. Expression levels of all three opsin genes increased significantly within the first two weeks of adulthood and following light exposure. Interestingly, the volumes of all three optic neuropils (lamina, medulla, and lobula) showed corresponding volume increases. Tracing of connections to higher visual centers in the mushroom bodies (MBs) revealed only one optic pathway, the anterior superior optic tract, emerging from the medulla and sending segregated input to the MB‐calyx collar. The MB collar volumes and densities of synaptic complexes (microglomeruli, MGs) increased with age. Exposure to light for 4 days induced a decrease in MG densities followed by an increase after extended light exposure. This shows that plasticity in retinal opsin gene expression and structural neuroplasticity in primary and secondary visual centers comprise both “experience‐independent” and “experience‐dependent” elements. We conclude that both sources of plasticity in the visual system represent important components promoting optimal timing of the interior–forager transition and flexibility of age‐related division of labor.

Evidence of a novel immune responsive protein in the Hymenoptera

Honeybee populations are severely threatened by parasites and diseases. Recent outbreaks of Colony Collapse Disorder (CCD) has caused loss of more than 35% of bee colonies in the USA, and this is thought to at least in part be due to parasites and/or disease. Interestingly, the honeybee possesses of a limited set of immune genes compared to other insects. Non-canonical immune genes of honeybee are of interest because they may provide greater insights into the peculiar nature of the immune system of this social insect. Previous analyses of bee haemolymph upon bacterial challenge identified a novel leucine-rich repeat protein termed IRP30. Here we show that IRP30 behaves as a typical secreted immune protein. It is expressed simultaneously with carboxylesterase upon treatment with bacteria or other elicitors of immune response. Furthermore we characterize the gene and the mRNA encoding this protein and the IRP30 protein itself. Its regulation and evolution reveal that IRP30 belongs to a protein family, distributed broadly among Hymenoptera, suggesting its ancient function in immune response. We document an interesting case of a recent IRP30 loss in the ant Atta cephalotes and hypothesize that a putative IRP30 homolog of Nasonia emerged by convergent evolution rather than diverged from a common ancestor.