Identification and characterisation of novel Mss4-binding Rab GTPases

The Mss4 (mammalian suppressor of yeast Sec4) is an evolutionarily highly conserved protein and is expressed in all mammalian tissues. Although its precise biological function is still elusive, it has been shown to associate with a subset of secretory Rab proteins (Rab1b, Rab3a, Rab8a, Rab10) and to possess a rather low guanine nucleotide exchange factor (GEF) activity towards them in vitro (Rab1, Rab3a and Rab8a). By screening a human placenta cDNA library with Mss4 as bait, we identified several Rab GTPases (Rab12, Rab13 and Rab18) as novel Mss4-binding Rab proteins. Only exocytic but no endocytic Rab GTPases were found in our search. The binding of Mss4 to Rab proteins was confirmed by direct yeast two-hybrid interaction, by co-immunoprecipitation from lysates of mammalian cells, by immunofluorescence colocalisation as well as by direct in vitro binding studies. Analysis of Mss4 catalytic activity towards different Rab substrates confirmed that it is a somewhat inefficient GEF. These data, together with our mutational analysis of Mss4-Rab binding capacity, support the already proposed idea that Mss4 functions rather as a chaperone for exocytic Rab GTPases than as a GEF.     

Translational thermotolerance provided by small heat shock proteins is limited to cap-dependent initiation and inhibited by 2-aminopurine

Heat shock results in inhibition of general protein synthesis. In thermotolerant cells, protein synthesis is still rapidly inhibited by heat stress, but protein synthesis recovers faster than in naive heat-shocked cells, a phenomenon known as translational thermotolerance. Here we investigate the effect of overexpressing a single heat shock protein on cap-dependent and cap-independent initiation of translation during recovery from a heat shock. When overexpressing alphaB-crystallin or Hsp27, cap-dependent initiation of translation was protected but no effect was seen on cap-independent initiation of translation. When Hsp70 was overexpressed however, both cap-dependent and -independent translation were protected. This finding indicates a difference in the mechanism of protection mediated by small or large heat shock proteins. Phosphorylation of alphaB-crystallin and Hsp27 is known to significantly decrease their chaperone activity; therefore, we tested phosphorylation mutants of these proteins in this system. AlphaB-crystallin needs to be in its non-phosphorylated state to give protection, whereas phosphorylated Hsp27 is more potent in protection than the unphosphorylatable form. This indicates that chaperone activity is not a prerequisite for protection of translation by small heat shock proteins after heat shock. Furthermore, we show that in the presence of 2-aminopurine, an inhibitor of kinases, among which is double-stranded RNA-activated kinase, the protective effect of overexpressing alphaB-crystallin is abolished. The synthesis of the endogenous Hsps induced by the heat shock to test for thermotolerance is also blocked by 2-aminopurine. Most likely the protective effect of alphaB-crystallin requires synthesis of the endogenous heat shock proteins. Translational thermotolerance would then be a co-operative effect of different heat shock proteins.     

Eukaryotic initiation factor 2alpha-independent pathway of stress granule induction by the natural product pateamine A

Stress granules are aggregates of small ribosomal subunits, mRNA, and numerous associated RNA-binding proteins that include several translation initiation factors. Stress granule assembly occurs in the cytoplasm of higher eukaryotic cells under a wide variety of stress conditions, including heat shock, UV irradiation, hypoxia, and exposure to arsenite. Thus far, a unifying principle of eukaryotic initiation factor 2alpha phosphorylation prior to stress granule formation has been observed from the majority of experimental evidence. Pateamine A, a natural product isolated from marine sponge, was recently reported to inhibit eukaryotic translation initiation and induce the formation of stress granules. In this report, the protein composition and fundamental progression of stress granule formation and disassembly induced by pateamine A was found to be similar to that for arsenite. However, pateamine A-induced stress granules were more stable and less prone to disassembly than those formed in the presence of arsenite. Most significantly, pateamine A induced stress granules independent of eukaryotic initiation factor 2alpha phosphorylation, suggesting an alternative mechanism of formation from that previously described for other cellular stresses. Taking into account the known inhibitory effect of pateamine A on eukaryotic translation initiation, a model is proposed to account for the induction of stress granules by pateamine A as well as other stress conditions through perturbation of any steps prior to the rejoining of the 60S ribosomal subunit during the entire translation initiation process.     

Thermal adaptation of the yeast mitochondrial Hsp70 system is regulated by the reversible unfolding of its nucleotide exchange factor

The Hsp70 protein switches during its functional cycle from an ADP-bound state with a high affinity for substrates to a low-affinity, ATP-bound state, with concomitant release of the client protein. The rate of the chaperone cycle is regulated by co-chaperones such as nucleotide exchange factors that significantly accelerate the ADP/ATP exchange. Mge1p, a mitochondrial matrix protein with homology to bacterial GrpE, serves as the nucleotide exchange factor of mitochondrial Hsp70. Here, we analyze the influence of temperature on the structure and functional properties of Mge1p from the yeast Saccharomyces cerevisiae. Mge1p is a dimer in solution that undergoes a reversible thermal transition at heat-shock temperatures, i.e. above 37 degrees C, that involves protein unfolding and dimer dissociation. The thermally denatured protein is unable to interact stably with mitochondrial Hsp70, and therefore is unable to regulate its ATPase and chaperone cycle. Crosslinking of wild-type mitochondria reveals that Mge1p undergoes the same dimer to monomer temperature-dependent shift, and that the nucleotide exchange factor does not associate with its Hsp70 partner at stress temperatures (i.e. > or =45 degrees C). Once the stress conditions disappear, Mge1p refolds and recovers both structure and functional properties. Therefore, Mge1p can act as a thermosensor for the mitochondrial Hsp70 system, regulating the nucleotide exchange rates under heat shock, as has been described for two bacterial GrpE proteins. The thermosensor activity is conserved in the GrpE-like nucleotide exchange factors although, as discussed here, it is achieved through a different structural mechanism.     

Mammalian stress granules represent sites of accumulation of stalled translation initiation complexes

In eukaryotic cells subjected to environmental stress, untranslated mRNA accumulates in discrete cytoplasmic foci that have been termed stress granules. Recent studies have shown that in addition to mRNA, stress granules also contain 40S ribosomal subunits and various translation initiation factors, including the mRNA binding proteins eIF4E and eIF4G. However, eIF2, the protein that transfers initiator methionyl-tRNA(i) (Met-tRNA(i)) to the 40S ribosomal subunit, has not been detected in stress granules. This result is surprising because the eIF2. GTP. Met-tRNA(i) complex is thought to bind to the 40S ribosomal subunit before the eIF4G. eIF4E. mRNA complex. In the present study, we show in both NIH-3T3 cells and mouse embryo fibroblasts that stress granules contain not only eIF2 but also the guanine nucleotide exchange factor for eIF2, eIF2B. Moreover, we show that phosphorylation of the alpha-subunit of eIF2 is necessary and sufficient for stress granule formation during the unfolded protein response. Finally, we also show that stress granules contain many, if not all, of the components of the 48S preinitiation complex, but not 60S ribosomal subunits, suggesting that they represent stalled translation initiation complexes.     

Structure of TCTP reveals unexpected relationship with guanine nucleotide-free chaperones

The translationally controlled tumor-associated proteins (TCTPs) are a highly conserved and abundantly expressed family of eukaryotic proteins that are implicated in both cell growth and the human acute allergic response but whose intracellular biochemical function has remained elusive. We report here the solution structure of the TCTP from Schizosaccharomyces pombe, which, on the basis of sequence homology, defines the fold of the entire family. We show that TCTPs form a structural superfamily with the Mss4/Dss4 family of proteins, which bind to the GDP/GTP free form of Rab proteins (members of the Ras superfamily) and have been termed guanine nucleotide-free chaperones (GFCs). Mss4 also acts as a relatively inefficient guanine nucleotide exchange factor (GEF). We further show that the Rab protein binding site on Mss4 coincides with the region of highest sequence conservation in the TCTP family. This is the first link to any other family of proteins that has been established for the TCTP family and suggests the presence of a GFC/GEF at extremely high abundance in eukaryotic cells.     

Metazoan Hsp70 machines use Hsp110 to power protein disaggregation

Accumulation of aggregation‐prone misfolded proteins disrupts normal cellular function and promotes ageing and disease. Bacteria, fungi and plants counteract this by solubilizing and refolding aggregated proteins via a powerful cytosolic ATP‐dependent bichaperone system, comprising the AAA+ disaggregase Hsp100 and the Hsp70‐Hsp40 system. Metazoa, however, lack Hsp100 disaggregases. We show that instead the Hsp110 member of the Hsp70 superfamily remodels the human Hsp70‐Hsp40 system to efficiently disaggregate and refold aggregates of heat and chemically denatured proteins in vitro and in cell extracts. This Hsp110 effect relies on nucleotide exchange, not on ATPase activity, implying ATP‐driven chaperoning is not required. Knock‐down of nematode Caenorhabditis elegans Hsp110, but not an unrelated nucleotide exchange factor, compromises dissolution of heat‐induced protein aggregates and severely shortens lifespan after heat shock. We conclude that in metazoa, Hsp70‐Hsp40 powered by Hsp110 nucleotide exchange represents the crucial disaggregation machinery that reestablishes protein homeostasis to counteract protein unfolding stress.     

Heat shock-induced translational alterations in HeLa cells. Initiation factor modifications and the inhibition of translation

Heat shock at 45 degrees C virtually abolishes protein synthesis in HeLa cells, but return to 37 degrees C effects a complete recovery and the concomitant synthesis of heat shock-induced proteins. Heat shock induces polysome disaggregation, indicating initiation is principally inhibited. In vitro assays for initiation factor activities reveal heat shock inhibits eukaryotic initiation factor 2 (eIF-2), eIF-(3 + 4F), and eIF-4B. Immunoblot analyses show that eIF-2 alpha and eIF-2 beta become modified during heat shock, and eIF-4B variants disappear. Upon return to 37 degrees C, these alterations reverse. The modifications of eIF-2 alpha and eIF-4B are due to phosphorylation and dephosphorylation, respectively. Enzymatic activities induced by heat shock inhibit protein synthesis and modify initiation factors in a rabbit reticulocyte lysate. Initiation factor modifications may contribute to, or cause, protein synthesis inhibition.     

Specific interactions of Mss4 with members of the Rab GTPase subfamily.

Mss4 is a mammalian protein that was identified as a suppressor of a yeast secretory mutant harboring a mutation in the GTPase Sec4 and was found to stimulate GDP release from this protein. We have now performed a biochemical characterization of the Mss4 protein and examined the specificity of its association with mammalian GTPases. Mss4 is primarily a soluble protein with a widespread tissue distribution. Recombinant Mss4 binds GTPases present in tissue extracts, and by a gel overlay assay binds specifically Rab Rab10proteins. We further define the Mss4-GTPase interaction to a subset of Rabs belonging to the same subfamily branch which include Rab1, Rab3, Rab8, Rab10, Sec4 and Ypt1 but not Rab2, Rab4, Rab5, Rab6, Rab9 and Rab11. Accordingly, Mss4 co-precipitates from a brain extract with Rab3a but not Rab5. Mss4 only stimulates GDP release from, and the association of GTP gamma S with, this Rab subset. Recombinant Mss4 and Rab3a form a stable complex in solution that is dissociated with either GDP or GTP gamma S. Injection of Mss4 into the squid giant nerve terminal enhances neurotransmitter release. These results suggest that Mss4 behaves as a guanylnucleotide exchange factor (GEF) for a subset of Rabs to influence distinct vesicular transport steps along the secretory pathway.     

Heat Shock-Induced Accumulation of Translation Elongation and Termination Factors Precedes Assembly of Stress Granules in S. cerevisiae

In response to severe environmental stresses eukaryotic cells shut down translation and accumulate components of the translational machinery in stress granules (SGs). Since they contain mainly mRNA, translation initiation factors and 40S ribosomal subunits, they have been referred to as dominant accumulations of stalled translation preinitiation complexes. Here we present evidence that the robust heat shock-induced SGs of S. cerevisiae also contain translation elongation factors eEF3 (Yef3p) and eEF1Bγ2 (Tef4p) as well as translation termination factors eRF1 (Sup45p) and eRF3 (Sup35p). Despite the presence of the yeast prion protein Sup35 in heat shock-induced SGs, we found out that its prion-like domain is not involved in the SGs assembly. Factors eEF3, eEF1Bγ2 and eRF1 were accumulated and co-localized with Dcp2 foci even upon a milder heat shock at 42°C independently of P-bodies scaffolding proteins. We also show that eEF3 accumulations at 42°C determine sites of the genuine SGs assembly at 46°C. We suggest that identification of translation elongation and termination factors in SGs might help to understand the mechanism of the eIF2α factor phosphorylation-independent repression of translation and SGs assembly.     

Poliovirus unlinks TIA1 aggregation and mRNA stress granule formation

In response to environmental stress and viral infection, mammalian cells form foci containing translationally silenced mRNPs termed stress granules (SGs). As aggregates of stalled initiation complexes, SGs are defined by the presence of translation initiation machinery in addition to mRNA binding proteins. Here, we report that cells infected with poliovirus (PV) can form SGs early that contain T-cell-restricted intracellular antigen 1 (TIA1), translation initiation factors, RNA binding proteins, and mRNA. However, this response is blocked as infection progresses, and a type of pseudo-stress granule remains at late times postinfection and contains TIA but lacks translation initiation factors, mRNA binding proteins, and most polyadenylated mRNA. This result was observed using multiple stressors, including viral infection, oxidative stress, heat shock, and endoplasmic reticulum stress. Multiple proteins required for efficient viral internal ribosome entry site-dependent translation are localized to SGs under stress conditions, providing a potential rationale for the evolution and maintenance of the SG inhibition phenotype. Further, the expression of a noncleavable form of the RasGAP-SH3 domain binding protein in PV-infected cells enables SGs whose constituents are consistent with the presence of stalled 48S translation preinitiation complexes to persist throughout infection. These results indicate that in poliovirus-infected cells, the functions of TIA self-aggregation and aggregation of stalled translation initiation complexes into stress granules are severed, leading to novel foci that contain TIA1 but lack other stress granule-defining components.     

Mechanism of Mss116 ATPase reveals functional diversity of DEAD-Box proteins

Mss116 is a Saccharomyces cerevisiae mitochondrial DEAD-box RNA helicase protein that is essential for efficient in vivo splicing of all group I and group II introns and for activation of mRNA translation. Catalysis of intron splicing by Mss116 is coupled to its ATPase activity. Knowledge of the kinetic pathway(s) and biochemical intermediates populated during RNA-stimulated Mss116 ATPase is fundamental for defining how Mss116 ATP utilization is linked to in vivo function. We therefore measured the rate and equilibrium constants underlying Mss116 ATP utilization and nucleotide-linked RNA binding. RNA accelerates the Mss116 steady-state ATPase ∼ 7-fold by promoting rate-limiting ATP hydrolysis such that inorganic phosphate (P_i) release becomes (partially) rate-limiting. RNA binding displays strong thermodynamic coupling to the chemical states of the Mss116-bound nucleotide such that Mss116 with bound ADP-P_i binds RNA more strongly than Mss116 with bound ADP or in the absence of nucleotide. The predominant biochemical intermediate populated during in vivo steady-state cycling is the strong RNA-binding Mss116-ADP-P_i state. Strong RNA binding allows Mss116 to fulfill its biological role in the stabilization of group II intron folding intermediates. ATPase cycling allows for transient population of the weak RNA-binding ADP state of Mss116 and linked dissociation from RNA, which is required for the final stages of intron folding. In cases where Mss116 functions as a helicase, the data collectively favor a model in which ATP hydrolysis promotes a weak-to-strong RNA binding transition that disrupts stable RNA duplexes. The subsequent strong-to-weak RNA binding transition associated with P_i release dissociates Mss116-RNA complexes, regenerating free Mss116.     

MRX8, the conserved mitochondrial YihA GTPase family member,is required for de novo Cox1 synthesis at suboptimal temperatures in Saccharomyces cerevisiae

The synthesis of Cox1, the conserved catalytic-core subunit of Complex IV, a multisubunit machinery of the mitochondrial oxidative phosphorylation (OXPHOS) system under environmental stress, has not been sufficiently addressed. In this study, we show that the putative YihA superfamily GTPase, Mrx8, is a bona fide mitochondrial protein required for Cox1 translation initiation and elongation during suboptimal growth condition at 16°C. Mrx8 was found in a complex with mitochondrial ribosomes, consistent with a role in protein synthesis. Cells expressing mutant Mrx8 predicted to be defective in guanine nucleotide binding and hydrolysis were compromised for robust cellular respiration. We show that the requirement of Pet309 and Mss51 for cellular respiration is not bypassed by overexpression of Mrx8 and vice versa. Consistently the ribosomal association of Mss51 is independent of Mrx8. Significantly, we find that GTPBP8, the human orthologue, complements the loss of cellular respiration in Δmrx8 cells and GTPBP8 localizes to the mitochondria in mammalian cells. This strongly suggests a universal role of the MRX8 family of proteins in regulating mitochondrial function.