Aggregation and Prion-Inducing Properties of the G-Protein Gamma Subunit Ste18 are Regulated by Membrane Association.

Yeast prions and mnemons are respectively transmissible and non-transmissible self-perpetuating protein assemblies, frequently based on cross-ß ordered detergent-resistant aggregates (amyloids). Prions cause devastating diseases in mammals and control heritable traits in yeast. It was shown that the de novo formation of the prion form [PSI+] of yeast release factor Sup35 is facilitated by aggregates of other proteins. Here we explore the mechanism of the promotion of [PSI+] formation by Ste18, an evolutionarily conserved gamma subunit of a G-protein coupled receptor, a key player in responses to extracellular stimuli. Ste18 forms detergent-resistant aggregates, some of which are colocalized with de novo generated Sup35 aggregates. Membrane association of Ste18 is required for both Ste18 aggregation and [PSI+] induction, while functional interactions involved in signal transduction are not essential for these processes. This emphasizes the significance of a specific location for the nucleation of protein aggregation. In contrast to typical prions, Ste18 aggregates do not show a pattern of heritability. Our finding that Ste18 levels are regulated by the ubiquitin-proteasome system, in conjunction with the previously reported increase in Ste18 levels upon the exposure to mating pheromone, suggests that the concentration-dependent Ste18 aggregation may mediate a mnemon-like response to physiological stimuli.

Role of the C-terminal basic amino acids and the lipid anchor of the Gγ2 protein in membrane interactions and cell localization.

Heterotrimeric G proteins are peripheral membrane proteins that frequently localize to the plasma membrane where their presence in molar excess over G protein coupled receptors permits signal amplification. Their distribution is regulated by protein-lipid interactions, which has a clear influence on their activity. Gßγ dimer drives the interaction between G protein heterotrimers with cell membranes. We focused our study on the role of the C-terminal region of the Gγ2 protein in G protein interactions with cell membranes. The Gγ2 subunit is modified at cysteine (Cys) 68 by the addition of an isoprenyl lipid, which is followed by the proteolytic removal of the last three residues that leaves an isoprenylated and carboxyl methylated Cys-68 as the terminal amino acid. The role of Cys isoprenylation of the CAAX box has been defined for other proteins, yet the importance of proteolysis and carboxyl methylation of isoprenylated proteins is less clear. Here, we showed that not only geranylgeranylation but also proteolysis and carboxyl methylation are essential for the correct localization of Gγ2 in the plasma membrane. Moreover, we showed the importance of electrostatic interactions between the inner leaflet of the plasma membrane and the positively charged C-terminal domain of the Gγ2 subunit (amino acids Arg-62, Lys-64 and Lys-65) as a second signal to reach the plasma membrane. Indeed, single or multiple point mutations at Gγ2 C-terminal amino acids have a significant effect on Gγ2 protein-plasma membrane interactions and its localization to charged Ld (liquid disordered) membrane microdomains. This article is part of a Special Issue entitled: Membrane Lipid Therapy: Drugs Targeting Biomembranes edited by Pablo V. Escribá.

Seipin mutation at glycosylation sites activates autophagy in transfected cells via abnormal large lipid droplets generation.

AIM: Seipin is a protein that resides in endoplasmic reticulum, and involved in both lipid metabolic disorders and motor neuropathy. The aim of this study was to investigate the effects of mutant seipin on autophagy system and the morphology of lipid droplets in vitro. METHODS: HEK-293, H1299 and MES23.5 cells were transfected with the plasmids of mutated seipin at glycosylation sites (N88S or S90L) and GFP-LC3 plasmids. The cells were subjected to immunofluorescence and flow cytometry assays, and the cell lysates were subjected to immunoblot analysis. Nile Red was used to stain the lipid droplets in the cells. RESULTS: Overexpression of the mutated seipin proteins N88S or S90L activated autophagy in the 3 cell lines, and substantially altered the sub-cellular distribution of the autophagosome marker GFP-LC3, leading to a number of large vacuoles appearing in the cytoplasm. The sub-cellular location of GFP-LC3 and mutated seipin proteins highly overlapped. Moreover, and the mutated seipin proteins caused diffuse small lipid droplets to fuse into larger lipid droplets. Treatment of mutated seipin-transfected cells with the autophagy inhibitor 3-MA (5 mmol/L) facilitated the fusion of mutated seipin-induced large vacuoles. The protein glycosylation inhibitor tunicamycin could mimic the mutated seipin-induced effects, and treatment of the wild-type seipin-transfected cells with tunicamycin (2.5 µg/mL) produced similar morphological and biochemical properties as in the mutated seipin-transfected cells. CONCLUSION: The mutation of seipin at glycosylation sites disrupt its function in regulating lipid droplet metabolism, and the autophagy acts as an adaptive response to break down abnormal lipid droplets. The interruption of autophagy would accelerate the fusion of abnormal lipid droplets.

Evidence for an unusual transmembrane configuration of AGG3, a class C Gγ subunit of Arabidopsis.

Heterotrimeric G proteins are crucial for the perception of external signals and subsequent signal transduction in animal and plant cells. In both model systems, the complex comprises one Gα, one Gß, and one Gγ subunit. However, in addition to the canonical Gγ subunits (class A), plants also possess two unusual, plant-specific classes of Gγ subunits (classes B and C) that have not yet been found in animals. These include Gγ subunits lacking the C-terminal CaaX motif (class B), which is important for membrane anchoring of the protein; the presence of such subunits gives rise to a flexible sub-population of Gß/γ heterodimers that are not necessarily restricted to the plasma membrane. Plants also contain class C Gγ subunits, which are twice the size of canonical Gγ subunits, with a predicted transmembrane domain and a large cysteine-rich extracellular C-terminus. However, neither the presence of the transmembrane domain nor the membrane topology have been unequivocally demonstrated. Here, we provide compelling evidence that AGG3, a class C Gγ subunit of Arabidopsis, contains a functional transmembrane domain, which is sufficient but not essential for plasma membrane localization, and that the cysteine-rich C-terminus is extracellular.

Characterization of inclusion bodies with cytoprotective properties formed by seipinopathy-linked mutant seipin.

Gain-of-toxic mutations in the N-glycosylation motif of the seipin/BSCL2 gene (namely, the N88S and S90L mutations) cause autosomal dominant motor neuron diseases, termed 'seipinopathy'. Expressed mutant seipin is improperly folded and accumulates in the endoplasmic reticulum (ER), leading to an unfolded protein response (UPR). Furthermore, cells expressing mutant seipin contain unique cytoplasmic inclusion bodies (IB) that form via a different mechanism from that of ubiquitinated inclusions, or aggresomes. Whether the formation of these IB is pathogenic or protective in neurodegenerative diseases remains unclear. Here, we determined that mutant seipin IB are negative for two well-established ER markers, immunoglobulin-heavy-chain-binding protein and calnexin, indicating a distinct compartmentalization from the main ER, and that mutant seipin IB are formed via a mechanism that is independent of major UPR transducers and ER chaperons. Electron microscopy and coexpression study with variant α1-antitrypsin cDNA showed that seipin IB are compatible with unique cytoplasmic vesicles known as ER-derived protective organelles (ERPO). We also obtained evidence that seipin IB exhibit a cytoprotective property via the attenuation of ER stress. These findings suggest that ERPO, such as seipin IB, are a novel adaptation machinery against the accumulation of unfolded proteins in the ER.

Multicolor BiFC analysis of G protein ßγ complex formation and localization.

Cells co-express multiple G protein ß and γ subunit isoforms, but the extent to which individual subunits associate to form particular ßγ complexes is not known. This issue is important because in vivo knockout experiments suggest that specific ßγ complexes may have unique functions despite the fact that most complexes exhibit similar properties when assayed in reconstituted systems. This chapter describes how multicolor bimolecular fluorescence complementation (BiFC) can be used in living cells to study the association preferences of ß and γ subunits. Multicolor BiFC determines the association preferences of these subunits by quantifying the two fluorescent complexes formed when ß or γ subunits fused to amino terminal fragments of yellow fluorescent protein (YFP-N) and cyan fluorescent protein (CFP-N) compete for interaction with limiting amounts of a common γ or ß subunit, respectively, fused to a carboxyl terminal fragment of CFP (CFP-C). One means by which ßγ complexes may differ from each other and thereby mediate unique functions in vivo is in the kinetics and patterns of their internalization responses to stimulation of G protein-coupled receptors (GPCRs). Methods are described for imaging and quantifying the internalization of pairs of ßγ complexes in response to GPCR stimulation in living cells.

Effect of phosducin silencing on the photokinetic motile response of Blepharisma japonicum.

The coloured ciliate Blepharisma japonicum changes swimming velocity (positive photokinesis) and elongates its body in response to a prolonged illumination. We have recently proposed that alterations in the phosphorylation level of the ciliate phosducin (Pdc) may be involved in light-induced cell elongation, which in turn affects the interaction of ßγ-dimer of G-proteins (Gßγ) with ß-tubulin and subsequent cytoskeletal remodelling. The cellular mechanism that governs the photokinetic effect in this ciliate has not been elucidated. In the present study, we utilise real-time PCR to demonstrate that the levels of ciliate Pdc mRNA are significantly reduced in Pdc-RNAi-treated cells compared to cells fed with bacteria carrying the empty vector (control cells). Using western immunoblotting, we confirmed that these cells treated with Pdc-RNAi expressed a substantially lower level of the Pdc protein. The assay also revealed that in ciliates treated with Pdc-RNAi and exposed to light, the cytosolic level of Gß (~36 kDa) was reduced, whereas the level of Gß localized to the membrane (~32 kDa) was increased compared to control cells. In addition, behavioural analysis of the cells indicated a substantial reduction of photokinesis. The findings in this study provide additional characterization of the functional properties of the ciliate Pdc protein and we discuss a likely role for this phosphoprotein in the photokinetic phenomenon of the ciliate protist Blepharisma.

Binding of ß4γ5 by adenosine A1 and A2A receptors determined by stable isotope labeling with amino acids in cell culture and mass spectrometry.

Characterization of G protein ßγ dimer isoform expression in different cellular contexts has been impeded by low levels of protein expression, broad isoform heterogeneity, and antibodies of limited specificity, sensitivity, or availability. As a new approach, we used quantitative mass spectrometry to characterize native ßγ dimers associated with adenosine A(1):α(i1) and adenosine A(2A):α(S) receptor fusion proteins expressed in HEK-293 cells. Cells expressing A(1):α(i1) were cultured in media containing [(13)C(6)]Arg and [(13)C(6)]Lys and ßγ labeled with heavy isotopes purified. Heavy ßγ was combined with either recombinant ßγ purified from Sf9 cells, ßγ purified from the A(2A):α(S) expressed in HEK-293 cells cultured in standard media, or an enriched ßγ fraction from HEK-293 cells. Samples were separated by SDS-PAGE, protein bands containing ß and γ were excised, digested with trypsin, and separated by HPLC, and isotope ratios were analyzed by mass spectrometry. Three ß isoforms, ß(1), ß(2), and ß(4), and seven γ isoforms, γ(2), γ(4), γ(5), γ(7), γ(10), γ(11), and γ(12), were identified in the analysis. ß(1) and γ(5) were most abundant in the enriched ßγ fraction, and this ßγ profile was generally mirrored in the fusion proteins. However, both A(2A):α(S) and A(1):α(i1) bound more ß(4) and γ(5) compared to the enriched ßγ fraction; also, more ß(4) was associated with A(2A):α(S) than A(1):α(i1). Both fusion proteins also contained less γ(2), γ(10), and γ(12) than the enriched ßγ fraction. These results suggest that preferences for particular ßγ isoforms may be driven in part by structural motifs common to adenosine receptor family members.

[BSCL2-related neurologic disorders/seipinopathy: endoplasmic reticulum stress in neurodegeneration].

The Seipin/BSCL2 gene was originally identified as a loss-of-function gene for congenital generalized lipodystrophy type 2, a condition characterized by severe lipoatrophy, insulin resistance, and hypertriglyceridemia. Whereas gain-of-toxic-function mutations (namely, mutations N88S and S90L) in the seipin gene have been identified in autosomal dominant motor neuron diseases such as Silver syndrome/spastic paraplegia 17 (SPG17) and distal hereditary motor neuropathy type V. Detailed phenotypic analyses have revealed that upper motor neurons, lower motor neurons and peripheral motor axons are variously affected in patients with these mutations. We recently showed that the N88S and S90L mutations disrupt the N-glycosylation motif, enhance ubiquitination, and appear to result in proteins that are improperly folded, leading to accumulation of the mutant protein in the endoplasmic reticulum (ER). We also showed that expression of mutant in cultured cells activates the UPR pathway and induces cell death, suggesting that seipinopathy is tightly associated with ER stress, which has recently been reported to be associated with other neurodegenerative diseases. Further study of the pathological mechanisms of the mutant forms of seipin may lead to important new insights into motor neuron diseases, including other spastic paraplegia diseases and amyotrophic lateral sclerosis.

Visualization of the interaction between Gbetagamma and tubulin during light-induced cell elongation of Blepharisma japonicum.

Blepharisma japonicum ciliates display reversible cell elongation in response to lasting bright illumination. This light-induced phenomenon has been ascribed to the active sliding of the cortical microtubules of the ciliate. The detailed intracellular signaling pathway that activates the microtubule network in response to light, resulting in cell elongation, is unknown. We have previously reported that light stimulation initiates sequential molecular events consisting of a decrease in the phosphorylation of ciliate Pdc, followed by increased binding of Pdc to membrane-localised Gbetagamma and the subsequent translocation of the Pdc-Gbetagamma complex to the cytoplasm. In this study, we used selected agents known to influence protein phosphorylation to test whether alterations in Pdc phosphorylation levels by light affect ciliate shape. Behavioural analysis indicated that cell treatment with okadaic acid, an inhibitor of protein phosphatase activity, heavily abolished the effect of light on cell elongation, whereas the presence of H-89, a specific inhibitor of cAMP-dependent protein kinase (PKA) activity, had no appreciable effect on the cell length. Phosphorylation assays showed that cell incubation with H-89 mimicked light by promoting Pdc dephosphorylation and its colocalization with Gbetagamma. However, as demonstrated by FRET-AP, Pdc-Gbetagamma complex formation and changes in the length of the cell did not occur under the same conditions. Moreover, fluorescence microscopy showed localization of Gbetagamma and beta-tubulin in the same cell compartment and demonstrated that a direct interaction between these proteins occurs in cells adapted to darkness or exposed to prolonged illumination (> or = 10 min). In contrast, an opposite effect, i.e. a transient decrease in the interaction between Gbetagamma and beta-tubulin and distinct Pdc dephosphorylation, was observed in cells illuminated for short time. Under these conditions, Pdc preferentially occupies the cell submembrane region and interacts with Gbetagamma. In cells illuminated for a longer time (> or = 10 min) and despite the constant light intensity, Pdc was progressively rephosphorylated and then dissociated from Gbetagamma, relocalizing within the cell cytoplasm. The results obtained in this study suggest that alterations in Pdc phosphorylation may be involved in light-induced elongation of the Blepharisma cell body, which affects the interaction of Gbetagamma with beta-tubulin and cell cytoskeleton remodelling.

Gbetagamma is a negative regulator of AP-1 mediated transcription.

Following stimulation of G protein-coupled receptors (GPCRs) at the cell surface, heterotrimeric G proteins are activated. Both Galpha and Gbetagamma subunits regulate specific effectors to transmit signals received by the receptor. Recent data suggest potential nuclear localization or translocation of the Gbetagamma subunit. Here, we show that co-expression of the Gbetagamma dimer decreased phorbol 12-myristate 13-acetate (PMA)-stimulated AP-1 gene reporter activity in HEK293 cells as well as the AP-1 dependent gonadotropin-releasing hormone-stimulated human follicle-stimulating hormone beta reporter activity in LbetaT2 gonadotrope cells. Further, we identify Fos transcription factors as novel interactors of the Gbeta1 subunit, using protein fragment complementation assays, as well as co-immunoprecipitation in vivo and in vitro. Fos proteins dimerize with Jun proteins to form activator protein-1 (AP-1) transcription factor complexes, which regulate target gene expression. Gbetagamma did not interfere with the dimerization of Fos and Jun or their ability to bind DNA. Rather, Gbetagamma co-localized with the AP-1 complex in the nucleus and recruited histone deacetylases (HDACs) to inhibit AP-1 transcriptional activity. Our data indicate a novel role for Gbetagamma subunits as transcriptional regulators.

Prenylation-deficient G protein gamma subunits disrupt GPCR signaling in the zebrafish.

Prenylation of G protein gamma (gamma) subunits is necessary for the membrane localization of heterotrimeric G proteins and for functional heterotrimeric G protein coupled receptor (GPCR) signaling. To evaluate GPCR signaling pathways during development, we injected zebrafish embryos with mRNAs encoding Ggamma subunits mutated so that they can no longer be prenylated. Low-level expression of these prenylation-deficient Ggamma subunits driven either ubiquitously or specifically in the primordial germ cells (PGCs) disrupts GPCR signaling and manifests as a PGC migration defect. This disruption results in a reduction of calcium accumulation in the protrusions of migrating PGCs and a failure of PGCs to directionally migrate. When co-expressed with a prenylation-deficient Ggamma, 8 of the 17 wildtype Ggamma isoforms individually confer the ability to restore calcium accumulation and directional migration. These results suggest that while the Ggamma subunits possess the ability to interact with G Beta (beta) proteins, only a subset of wildtype Ggamma proteins are stable within PGCs and can interact with key signaling components necessary for PGC migration. This in vivo study highlights the functional redundancy of these signaling components and demonstrates that prenylation-deficient Ggamma subunits are an effective tool to investigate the roles of GPCR signaling events during vertebrate development.

Characterization of seipin/BSCL2, a protein associated with spastic paraplegia 17.

Seipin, which is encoded by the BSCL2 gene, is a glycoprotein of unknown biochemical function that is associated with dominant hereditary motor neuron diseases. Mutations in the N-glycosylation site of seipin are associated with the disease states and result in accumulation of unfolded protein in the endoplasmic reticulum (ER), leading to the unfolded protein response (UPR) and cell death, suggesting that these diseases are tightly associated with ER stress. Here, we determined the subcellular localization, functional domains, and distribution of seipin in tissues. Our studies show that the transmembrane domains in seipin are critical for ER retention, ubiquitination, formation of inclusions, and activation of UPR. Using immunohistochemistry, seipin expression is detected in neurons in the spinal cord and in the frontal lobe cortex of the brain. The present study provides new insights into the biology of seipin protein that should help our understanding of the pathogenesis of seipin-related diseases.

G protein betagamma subunits interact with alphabeta- and gamma-tubulin and play a role in microtubule assembly in PC12 cells.

The betagamma subunit of G proteins (Gbetagamma) is known to transfer signals from cell surface receptors to intracellular effector molecules. Recent results suggest that Gbetagamma also interacts with microtubules and is involved in the regulation of the mitotic spindle. In the current study, the anti-microtubular drug nocodazole was employed to investigate the mechanism by which Gbetagamma interacts with tubulin and its possible implications in microtubule assembly in cultured PC12 cells. Nocodazole-induced depolymerization of microtubules drastically inhibited the interaction between Gbetagamma and tubulin. Gbetagamma was preferentially bound to microtubules and treatment with nocodazole suggested that the dissociation of Gbetagamma from microtubules is an early step in the depolymerization process. When microtubules were allowed to recover after removal of nocodazole, the tubulin-Gbetagamma interaction was restored. Unlike Gbetagamma, however, the interaction between tubulin and the alpha subunit of the Gs protein (Gsalpha) was not inhibited by nocodazole, indicating that the inhibition of tubulin-Gbetagamma interactions during microtubule depolymerization is selective. We found that Gbetagamma also interacts with gamma-tubulin, colocalizes with gamma-tubulin in centrosomes, and co-sediments in centrosomal fractions. The interaction between Gbetagamma and gamma-tubulin was unaffected by nocodazole, suggesting that the Gbetagamma-gamma-tubulin interaction is not dependent on assembled microtubules. Taken together, our results suggest that Gbetagamma may play an important and definitive role in microtubule assembly and/or stability. We propose that betagamma-microtubule interaction is an important step for G protein-mediated cell activation. These results may also provide new insights into the mechanism of action of anti-microtubule drugs.

Dual lipid modification of Arabidopsis Ggamma-subunits is required for efficient plasma membrane targeting.

Posttranslational lipid modifications are important for proper localization of many proteins in eukaryotic cells. However, the functional interrelationships between lipid modification processes in plants remain unclear. Here we demonstrate that the two heterotrimeric G-protein gamma-subunits from Arabidopsis (Arabidopsis thaliana), AGG1 and AGG2, are prenylated, and AGG2 is S-acylated. In wild type, enhanced yellow fluorescent protein-fused AGG1 and AGG2 are associated with plasma membranes, with AGG1 associated with internal membranes as well. Both can be prenylated by either protein geranylgeranyltransferase I (PGGT-I) or protein farnesyltransferase (PFT). Their membrane localization is intact in mutants lacking PFT activity and largely intact in mutants lacking PGGT-I activity but is disrupted in mutants lacking both PFT and PGGT-I activity. Unlike in mammals, Arabidopsis Ggammas do not rely on functional Galpha for membrane targeting. Mutation of the sixth to last cysteine, the putative S-acylation acceptor site, causes a dramatic change in AGG2 but not AGG1 localization pattern, suggesting S-acylation serves as an important additional signal for AGG2 to be targeted to the plasma membrane. Domain-swapping experiments suggest that a short charged sequence at the AGG2 C terminus contributes to AGG2's efficient membrane targeting compared to AGG1. Our data show the large degree to which PFT and PGGT-I can compensate for each other in plants and suggest that differential lipid modification plays an important regulatory role in plant protein localization.

Immunohistochemical localization of G protein betagamma subunits in the lateral wall of the rat cochlea.

The role of G protein-mediated signal transduction in the production of endolymph, an extracellular fluid of unusual ionic composition, is beginning to be understood. The identity of Galpha subunits in the stria vascularis and the spiral ligament of the lateral wall of the cochlear duct is well established. However, little is known about the presence of betagamma subunits. This study used immunohistochemistry to investigate the distribution of G protein betagamma subunits in the lateral wall of the cochlea. Temporal bones of 6- to 8-week-old rats were fixed in 4% paraformaldehyde and 0.1% glutaraldehyde and processed for embedding in paraffin wax. The dewaxed, midmodiolar sections of the cochlea were incubated with subunit-specific polyclonal antibodies. The results show that the pattern of immunoreactivity varies for the G protein beta1-4 and gamma1-3, 5 and 7 subunits in the stria vascularis and spiral ligament. In the stria vascularis, immunoreactivity was detected for beta2, beta3, beta4, gamma1, gamma2 and gamma7 subunits. All five types of fibrocytes in the spiral ligament exhibited positive staining for gamma2 and gamma7. However, immunoreactivity for beta1-4 subunits was variable. Immunoreactivity for gamma3 and gamma5 subunits was not detected in the lateral cochlear wall. The expression pattern of G protein betagamma subunits in lateral wall provides a basis for interpreting the functions of G protein-coupled receptors in cochlear fluid homeostasis.

The phosducin-like protein PhLP1 is essential for G{beta}{gamma} dimer formation in Dictyostelium discoideum.

Phosducin proteins are known to inhibit G protein-mediated signaling by sequestering Gbetagamma subunits. However, Dictyostelium discoideum cells lacking the phosducin-like protein PhLP1 display defective rather than enhanced G protein signaling. Here we show that green fluorescent protein (GFP)-tagged Gbeta (GFP-Gbeta) and GFP-Ggamma subunits exhibit drastically reduced steady-state levels and are absent from the plasma membrane in phlp1(-) cells. Triton X-114 partitioning suggests that lipid attachment to GFP-Ggamma occurs in wild-type cells but not in phlp1(-) and gbeta(-) cells. Moreover, Gbetagamma dimers could not be detected in vitro in coimmunoprecipitation assays with phlp1(-) cell lysates. Accordingly, in vivo diffusion measurements using fluorescence correlation spectroscopy showed that while GFP-Ggamma proteins are present in a complex in wild-type cells, they are free in phlp1(-) and gbeta(-) cells. Collectively, our data strongly suggest the absence of Gbetagamma dimer formation in Dictyostelium cells lacking PhLP1. We propose that PhLP1 serves as a cochaperone assisting the assembly of Gbeta and Ggamma into a functional Gbetagamma complex. Thus, phosducin family proteins may fulfill hitherto unsuspected biosynthetic functions.