Interaction Of Glyceraldehyde-3-phosphate Dehydrogenase Research Articles

Biochemical evidence that the whole compartment activity behavior of GAPDH differs between the cytoplasm and nucleus.

Some metabolic enzymes normally occur in the nucleus and cytoplasm. These compartments differ in molecular composition. Since post-translational modification and interaction with allosteric effectors can tune enzyme activity, it follows that the behavior of an enzyme as a catalyst may differ between the cytoplasm and nucleus. We explored this possibility for the glycolytic enzyme glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Homogenates of pristine nuclei and cytoplasms isolated from Xenopus laevis oocytes were used for whole compartment activity profiling in a near-physiological buffer. Titrations of NAD+ revealed similar whole compartment activity profiles for GAPDH in nuclear and cytoplasmic homogenates. Surprisingly however GAPDH in these compartments did not have the same behavior in assays of the dependence of initial velocity (v0) on G3P concentration. First, the peak v0 for nuclear GAPDH was up to 2.5-fold higher than the peak for cytoplasmic GAPDH. Second, while Michaelis Menten-like behavior was observed in all assays of cytoplasm, the v0 versus [G3P] plots for nuclear GAPDH typically exhibited a non-Michaelis Menten (sigmoidal) profile. Apparent Km and Vmax (G3P) values for nuclear GAPDH activity were highly variable, even between replicates of the same sample. Possible sources of this variability include in vitro processing of a metabolite that allosterically regulates GAPDH, turnover of a post-translational modification of the enzyme, and fluctuation of the state of interaction of GAPDH with other proteins. Collectively these findings are consistent with the hypothesis that the environment of the nucleus is distinct from the environment of the cytoplasm with regard to GAPDH activity and its modulation. This finding warrants further comparison of the regulation of nuclear and cytoplasmic GAPDH, as well as whole compartment activity profiling of other enzymes of metabolism with cytosolic and nuclear pools.

Hydrogen sulfide-induced GAPDH sulfhydration disrupts the CCAR2-SIRT1 interaction to initiate autophagy

ABSTRACT The deacetylase SIRT1 (sirtuin 1) has emerged as a major regulator of nucleocytoplasmic distribution of macroautophagy/autophagy marker MAP1LC3/LC3 (microtubule-associated protein 1 light chain 3). Activation of SIRT1 leads to the deacetylation of LC3 and its translocation from the nucleus into the cytoplasm leading to an increase in the autophagy flux. Notably, hydrogen sulfide (H2S) is a cytoprotective gasotransmitter known to activate SIRT1 and autophagy; however, the underlying mechanism for both remains unknown. Herein, we demonstrate that H2S sulfhydrates the active site cysteine of the glycolytic enzyme GAPDH (glyceraldehyde-3-phosphate dehydrogenase). Sulfhydration of GAPDH leads to its redistribution into the nucleus. Importantly, nuclear localization of GAPDH is critical for H2S-mediated activation of autophagy as H2S does not induce autophagy in cells with GAPDH ablation or cells overexpressing a GAPDH mutant lacking the active site cysteine. Importantly, we observed that nuclear GAPDH interacts with CCAR2/DBC1 (cell cycle activator a nd apoptosis regulator 2) inside the nucleus. CCAR2 interacts with the deacetylase SIRT1 to inhibit its activity. Interaction of GAPDH with CCAR2 disrupts the inhibitory effect of CCAR2 on SIRT1. Activated SIRT1 then deacetylates MAP1LC3B/LC3B (microtubule-associated protein 1 light chain 3 beta) to induce its translocation into the cytoplasm and activate autophagy. Additionally, we demonstrate this pathway’s physiological role in autophagy-mediated trafficking of Mycobacterium tuberculosis into lysosomes to restrict intracellular mycobacteria growth. We think that the pathway described here could be involved in H2S-mediated clearance of intracellular pathogens and other health benefits. Abbreviations: ATG5: autophagy related 5; ATG7: autophagy related 7; BECN1: beclin 1, autophagy related; CCAR2/DBC1: cell cycle activator and apoptosis regulator 2; CFU: colony-forming units; DLG4/PSD95: discs large MAGUK scaffold protein 4; EX-527: 6-chloro-2,3,4,9-tetrahydro-1H-carbazole-1-carboxamide; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; H2S: hydrogen sulfide; HEK: human embryonic kidney cells; MAP1LC3B/LC3B: microtubule-associated protein 1 light chain 3 beta; MEF: mouse embryonic fibroblast; Mtb: Mycobacterium tuberculosis; MTOR: mechanistic target of rapamycin kinase; MOI: multiplicity of infection; NO: nitric oxide; PI3K: phosphatidylinositol-4,5-bisphosphate 3-kinase; PLA: proximity ligation assay; PRKAA: protein kinase, AMP-activated, alpha catalytic subunit; SIAH1: siah E3 ubiquitin protein ligase 1A; SIRT1: sirtuin 1; TB: tuberculosis; TP53INP2/DOR: transformation related protein 53 inducible nuclear protein 2; TRP53/TP53: transformation related protein 53

Glycation of glyceraldehyde-3-phosphate dehydrogenase inhibits the binding with α-synuclein and RNA

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) shows great diversity of functions, interaction partners and post-translational modifications. GAPDH undergoes glycation of positively charged residues in diabetic patient's tissues and therefore may change interaction with partners. The influence of GAPDH glycation on interaction with two important partners, α-synuclein and RNA, has been investigated in silico using molecular dynamics simulations and in vitro using surface plasmon resonance measurements. Since positively charged groove including substrate- and NAD+-binding sites is proposed as potential binding site for α-synuclein and RNA, GAPDH was glycated on residues in grooves and randomly distributed over the whole surface. Lysine residues were replaced with negatively charged carboxymethyl lysine as a widespread advanced glycation end product. As results, GAPDH glycation suppressed the interaction with α-synuclein and RNA. Although the modified GAPDH residues participated in binding with α-synuclein, no stable binding site with both glycated forms was observed. Glycation along the whole GAPDH surface completely suppressed interaction with RNA, whereas the alternative possible RNA binding site was identified in case of groove glycation. The findings were supported by direct measurement of the binding affinity. The obtained results clarify effect of glycation on GAPDH interaction with α-synuclein and RNA and elucidate a possible mechanism of interplay between glycation occurred in diabetes and neurodegenerative diseases, which GAPDH and α-synuclein are involved in.

A ‘sweet spot’ for complement receptor CD46 activity via direct regulation of glycolytic vs. moonlighting functions of GAPDH

Abstract Autocrine engagement of the complement receptor CD46, driven by T cell receptor (TCR) activation on human CD4+ T cells, is critical for human Th1 induction: Processing of the intracellular CD46 domains by γ-secretase and their nuclear translocation mediates the expression of nutrient transporters GLUT1 and LAT1, increases glycolysis and oxidative phosphorylation, and enables metabolic reprogramming required for cell growth, expansion and effector function. How, at the molecular level, CYT-1 enables this fundamental cellular adaptation remains unknown. To probe for CYT-1 interacting proteins we generated antibodies against cleaved CYT-1 and performed co-immunoprecipitation experiments. Unexpectedly, mass spectrometry revealed association of CYT-1 with several glycolytic enzymes, including glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Although ELISA and MST-based experiments confirmed a strong, dose-dependent CYT-1/GAPDH interaction, glycolytic activity was hardly affected by CYT-1. Rather, CYT-1 modulated the activity of a key ‘moonlighting function’ of GAPDH, namely the posttranscriptional regulation of mRNA stability/translation: CYT-1 binding fostered desoligomerization of glycolytically active GAPDH tetramers to monomers with altered mRNA binding abilities – particularly for mRNAs encoding metabolic enzymes. These data identify CD46 as a ‘shape-shifter’ of GAPDH activity, critically balancing its canonical (glycolysis) versus non-canonical (posttranscriptional regulation) activity – further substantiating the notion that autocrine complement is a critical regulator of normal cell physiology.

Role of Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) in DNA Repair.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is widely known as a glycolytic enzyme. Nevertheless, various functions of GAPDH have been found that are unrelated to glycolysis. Some of these functions presume interaction of GAPDH with DNA, but the mechanism of its translocation to the nucleus is not fully understood. When in the nucleus, GAPDH participates in the initiation of apoptosis and transcription of genes involved in antiapoptotic pathways and cell proliferation and plays a role in the regulation of telomere length. Several authors have shown that GAPDH displays the uracil-DNA glycosylase activity and interacts with some types of DNA damages, such as apurinic/apyrimidinic sites, nucleotide analogs, and covalent DNA adducts with alkylating agents. Moreover, GAPDH can interact with proteins participating in DNA repair, such as APE1, PARP1, HMGB1, and HMGB2. In this review, the functions of GAPDH associated with DNA repair are discussed in detail.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) interacts with apurinic/apyrimidinic sites in DNA

Apurinic/apyrimidinic (AP) sites are some of the most frequent DNA damages and the key intermediates of base excision repair. Certain proteins can interact with the deoxyribose of the AP site to form a Schiff base, which can be stabilized by NaBH4 treatment. Several types of DNA containing the AP site were used to trap proteins in human cell extracts by this method. In the case of single-stranded AP DNA and AP DNA duplex with both 5′ and 3′ dangling ends, the major crosslinking product had an apparent molecular mass of 45kDa. Using peptide mass mapping based on mass spectrometry data, we identified the protein forming this adduct as an isoform of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) called “uracil-DNA glycosylase”. GAPDH is a glycolytic enzyme with many additional putative functions, which include interaction with nucleic acids, different DNA damages and DNA repair enzymes. We investigated interaction of GAPDH purified from HeLa cells and rabbit muscles with different AP DNAs. In spite of the ability to form a Schiff-base intermediate with the deoxyribose of the AP site, GAPDH does not display the AP lyase activity. In addition, along with the borohydride-dependent adducts with AP DNAs containing single-stranded regions, GAPDH was also shown to form the stable borohydride-independent crosslinks with these AP DNAs. GAPDH was proven to crosslink preferentially to AP DNAs cleaved via the β-elimination mechanism (spontaneously or by AP lyases) as compared to DNAs containing the intact AP site. The level of GAPDH–AP DNA adduct formation depends on oxidation of the protein SH-groups; disulfide bond reduction in GAPDH leads to the loss of its ability to form the adducts with AP DNA. A possible role of formation of the stable adducts with AP sites by GAPDH is discussed.

Glyceraldehyde-3-phosphate dehydrogenase is required for efficient repair of cytotoxic DNA lesions in Escherichia coli

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a multifunctional protein with diverse biological functions in human cells. In bacteria, moonlighting GAPDH functions have only been described for the secreted protein in pathogens or probiotics. At the intracellular level, we previously reported the interaction of Escherichia coli GAPDH with phosphoglycolate phosphatase, a protein involved in the metabolism of the DNA repair product 2-phosphoglycolate, thus suggesting a putative role of GAPDH in DNA repair processes. Here, we provide evidence that GAPDH is required for the efficient repair of DNA lesions in E. coli. We show that GAPDH-deficient cells are more sensitive to bleomycin or methyl methanesulfonate. In cells challenged with these genotoxic agents, GAPDH deficiency results in reduced cell viability and filamentous growth. In addition, the gapA knockout mutant accumulates a higher number of spontaneous abasic sites and displays higher spontaneous mutation frequencies than the parental strain. Pull-down experiments in different genetic backgrounds show interaction between GAPDH and enzymes of the base excision repair pathway, namely the AP-endonuclease Endo IV and uracil DNA glycosylase. This finding suggests that GAPDH is a component of a protein complex dedicated to the maintenance of genomic DNA integrity. Our results also show interaction of GAPDH with the single-stranded DNA binding protein. This interaction may recruit GAPDH to the repair sites and implicates GAPDH in DNA repair pathways activated by profuse DNA damage, such as homologous recombination or the SOS response.

Glyceraldehyde-3-phosphate dehydrogenase acts as a mitochondrial trans-S-nitrosylase in the heart.

Mitochondrial proteins have been shown to be common targets of S-nitrosylation (SNO), but the existence of a mitochondrial source of nitric oxide remains controversial. SNO is a nitric oxide-dependent thiol modification that can regulate protein function. Interestingly, trans-S-nitrosylation represents a potential pathway for the import of SNO into the mitochondria. The glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which has been shown to act as a nuclear trans-S-nitrosylase, has also been shown to enter mitochondria. However, the function of GAPDH in the mitochondria remains unknown. Therefore, we propose the hypothesis that S-nitrosylated GAPDH (SNO-GAPDH) interacts with mitochondrial proteins as a trans-S-nitrosylase. In accordance with this hypothesis, SNO-GAPDH should be detected in mitochondrial fractions, interact with mitochondrial proteins, and increase mitochondrial SNO levels. Our results demonstrate a four-fold increase in GAPDH levels in the mitochondrial fraction of mouse hearts subjected to ischemic preconditioning, which increases SNO-GAPDH levels. Co-immunoprecipitation studies performed in mouse hearts perfused with the S-nitrosylating agent S-nitrosoglutathione (GSNO), suggest that SNO promotes the interaction of GAPDH with mitochondrial protein targets. The addition of purified SNO-GAPDH to isolated mouse heart mitochondria demonstrated the ability of SNO-GAPDH to enter the mitochondrial matrix, and to increase SNO for many mitochondrial proteins. Further, the overexpression of GAPDH in HepG2 cells increased SNO for a number of different mitochondrial proteins, including heat shock protein 60, voltage-dependent anion channel 1, and acetyl-CoA acetyltransferase, thus supporting the role of GAPDH as a potential mitochondrial trans-S-nitrosylase. In further support of this hypothesis, many of the mitochondrial SNO proteins identified with GAPDH overexpression were no longer detected with GAPDH knock-down or mutation. Therefore, our results suggest that SNO-GAPDH can act as a mitochondrial trans-S-nitrosylase, thereby conferring the transfer of SNO from the cytosol to the mitochondria.

GAPDH: the missing link between glycolysis and mitochondrial oxidative phosphorylation?

The main function of glycolysis and oxidative phosphorylation is to produce cellular energy in the form of ATP. In the present paper we propose a link between both of these energy-regulatory processes in the form of GAPDH (glyceraldehyde-3-phosphate dehydrogenase) and CytOx (cytochrome c oxidase). GAPDH is the sixth enzyme of glycolysis, whereas CytOx is the fourth complex of the mitochondrial oxidative phosphorylation system. In MS analysis, GAPDH was found to be associated with a BN-PAGE (blue native PAGE)-isolated complex of CytOx from bovine heart tissue homogenates. Both GAPDH and CytOx are highly regulated under normal energy metabolic conditions, but both of these enzymes are highly deregulated in the presence of oxidative stress. The interaction of GAPDH with CytOx could be the point of interest as it has already been shown that GAPDH protein damage results in a marked decrease in cellular ATP levels. On the other hand, decreasing the ATP/ADP ratio may ultimately result in switching off the allosteric ATP inhibition of CytOx leading to increased ROS (reactive oxygen species), cytochrome c release and apoptosis. Moreover, we have previously reported that allosteric ATP inhibition of CytOx is responsible for keeping the membrane potential at low healthy values, thus avoiding the production of ROS and this allosteric ATP inhibition is switched on at a high ATP/ADP ratio. So, in the present paper, we propose a scheme that could prove to be a link between these two enzymes and their role in the prevalence of diseases.

Glyceraldehyde-3-Phosphate Dehydrogenase Interacts with Proapoptotic Kinase Mst1 to Promote Cardiomyocyte Apoptosis

Mammalian sterile 20-like kinase 1 (Mst1) is a critical component of the Hippo signaling pathway, which regulates a variety of biological processes ranging from cell contact inhibition, organ size control, apoptosis and tumor suppression in mammals. Mst1 plays essential roles in the heart disease since its activation causes cardiomyocyte apoptosis and dilated cardiomyopathy. However, the mechanism underlying Mst1 activation in the heart remains unknown. In a yeast two-hybrid screen of a human heart cDNA library with Mst1 as bait, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was identified as an Mst1-interacting protein. The interaction of GAPDH with Mst1 was confirmed by co-immunoprecipitation in both co-transfected HEK293 cells and mouse heart homogenates, in which GAPDH interacted with the kinase domain of Mst1, whereas the C-terminal catalytic domain of GAPDH mediated its interaction with Mst1. Moreover, interaction of Mst1 with GAPDH caused a robust phosphorylation of GAPDH and markedly increased the Mst1 activity in cells. Chelerythrine, a potent inducer of apoptosis, substantially increased the nuclear translocation and interaction of GAPDH and Mst1 in cardiomyocytes. Overexpression of GAPDH significantly augmented the Mst1 mediated apoptosis, whereas knockdown of GAPDH markedly attenuated the Mst1 activation and cardiomyocyte apoptosis in response to either chelerythrine or hypoxia/reoxygenation. These findings reveal a novel function of GAPDH in Mst1 activation and cardiomyocyte apoptosis and suggest that disruption of GAPDH interaction with Mst1 may prevent apoptosis related heart diseases such as heart failure and ischemic heart disease.

Protein‐Polyelectrolyte Complexes. How to Suppress Thermoaggregation without Noticeable Denaturing of the Protein?

AbstractHomotetrameric enzyme glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) was used as a model protein for elucidation a capability of the oppositely charged polyelectrolyte to suppress the protein thermoaggregation. Charge density and degree of polymerization of the chains, relative content of GAPDH in the mixture, as well as pH and ionic strength proved to be the factors of the thermoaggregation control. However the electrostatic binding of the most active suppressors, e.g. sodium poly(styrenesulfonate) was attended with marked changes of secondary, tertiary and quaternary structure of GAPDH and clearly defined denaturing. The performed comprehensive study of GAPDH interaction with different tightly bound polysulfoanions revealed the key role of a degree of polymerization and hydrophobicity of the chains in the denaturing, specifically the pronounced adverse effect on the enzyme of relatively hydrophobic short‐chained polyanions. Accordingly, the required criteria have been stated as follows: relatively hydrophilic highly polymerized polyelectrolytes that are able to form with a protein of fairly stable water‐soluble complexes are best suited to prevent thermoaggregation without drastic change of the protein structure.

Glyceraldehyde 3-Phosphate Dehydrogenase Negatively Regulates the Replication of Bamboo Mosaic Virus and Its Associated Satellite RNA

The identification of cellular proteins associated with virus replicase complexes is crucial to our understanding of virus-host interactions, influencing the host range, replication, and virulence of viruses. A previous in vitro study has demonstrated that partially purified Bamboo mosaic virus (BaMV) replicase complexes can be employed for the replication of both BaMV genomic and satellite BaMV (satBaMV) RNAs. In this study, we investigated the BaMV and satBaMV 3' untranslated region (UTR) binding proteins associated with these replicase complexes. Two cellular proteins with molecular masses of ∼35 and ∼55 kDa were specifically cross-linked with RNA elements, whereupon the ∼35-kDa protein was identified as the glycolytic enzyme glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Gel mobility shift assays confirmed the direct interaction of GAPDH with the 3' UTR sequences, and competition gel shift analysis revealed that GAPDH binds preferentially to the positive-strand BaMV and satBaMV RNAs over the negative-strand RNAs. It was observed that the GAPDH protein binds to the pseudoknot poly(A) tail of BaMV and stem-loop-C poly(A) tail of satBaMV 3' UTR RNAs. It is important to note that knockdown of GAPDH in Nicotiana benthamiana enhances the accumulation of BaMV and satBaMV RNA; conversely, transient overexpression of GAPDH reduces the accumulation of BaMV and satBaMV RNA. The recombinant GAPDH principally inhibits the synthesis of negative-strand RNA in exogenous RdRp assays. These observations support the contention that cytosolic GAPDH participates in the negative regulation of BaMV and satBaMV RNA replication.

Human Glyceraldehyde-3-phosphate Dehydrogenase Plays a Direct Role in Reactivating Oxidized Forms of the DNA Repair Enzyme APE1

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has diverse biological functions including its nuclear translocation in response to oxidative stress. We show that GAPDH physically associates with APE1, an essential enzyme involved in the repair of abasic sites in damaged DNA, as well as in the redox regulation of several transcription factors. This interaction allows GAPDH to convert the oxidized species of APE1 to the reduced form, thereby reactivating its endonuclease activity to cleave abasic sites. The GAPDH variants C152G and C156G retain the ability to interact with but are unable to reactivate APE1, implicating these cysteines in catalyzing the reduction of APE1. Interestingly, GAPDH-small interfering RNA knockdown sensitized the cells to methyl methane sulfonate and bleomycin, which generate lesions that are repaired by APE1, but showed normal sensitivity to 254-nm UV. Moreover, the GAPDH knockdown cells exhibited an increased level of spontaneous abasic sites in the genomic DNA as a result of diminished APE1 endonuclease activity. Thus, the nuclear translocation of GAPDH during oxidative stress constitutes a protective mechanism to safeguard the genome by preventing structural inactivation of APE1.

Glyceraldehyde 3-phosphate dehydrogenase is a SET-binding protein and regulates cyclin B-cdk1 activity

We report here that glyceraldehyde-3-phosphate dehydrogenase (GAPDH) interacts in vitro and in vivo with the protein SET. This interaction is performed through the acidic domain of SET located at the carboxy terminal region. On analysing the functional relevance of SET-GAPDH interaction, we observed that GAPDH reverses in a dose-dependent manner, the inhibition of cyclin B-cdk1 activity produced by SET. Similarly to SET, GAPDH associates with cyclin B, suggesting that the regulation of cyclin B-cdk1 activity might be mediated not only by the interaction of GAPDH with SET but also with cyclin B. To analyse the putative role of GAPDH on cell cycle progression, HCT116 cells were transfected with a GAPDH expression vector. Results indicate that overexpression of GAPDH does not affect the timing of DNA replication but induces an increase in the number of mitosis, an advancement of the peak of cyclin B-cdk1 activity and an acceleration of cell cycle progression. All these results suggest that GAPDH might be involved in cell cycle regulation by modulating cyclin B-cdk1 activity.

GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE, APOPTOSIS, AND NEURODEGENERATIVE DISEASES

Increasing evidence supports the notion that glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a protein with multiple functions, including its surprising role in apoptosis. GAPDH is overexpressed and accumulates in the nucleus during apoptosis induced by a variety of insults in diverse cell types. Knockdown of GAPDH using an antisense strategy demonstrates its involvement in the apoptotic cascade in which GAPDH nuclear translocation appears essential. Knowledge concerning the mechanisms underlying GAPDH nuclear translocation and subsequent cell death is growing. Additional evidence suggests that GAPDH may be an intracellular sensor of oxidative stress during early apoptosis. Abnormal expression, nuclear accumulation, changes in physical properties, and loss of glycolytic activity of GAPDH have been found in cellular and transgenic models as well as postmortem tissues of several neurodegenerative diseases. The interaction of GAPDH with disease-related proteins as well as drugs used to treat these diseases suggests that it is a potential molecular target for drug development.

Glyceraldehyde-3-phosphate Dehydrogenase Interacts with Rab2 and Plays an Essential Role in Endoplasmic Reticulum to Golgi Transport Exclusive of Its Glycolytic Activity

Rab2 requires atypical protein kinase C iota/lambda (aPKC iota/lambda) to promote vesicle formation from vesicular tubular clusters (VTCs). The Rab2-generated vesicles are enriched in recycling proteins suggesting that the carriers are retrograde-directed and retrieve transport machinery back to the endoplasmic reticulum. These vesicles also contained the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH). We have previously established that GAPDH is required for membrane transport between the endoplasmic reticulum and the Golgi complex. Moreover, GAPDH is phosphorylated by aPKC iota/lambda and binds to the aPKC iota/lambda regulatory domain. In this study, we employed a combination of in vivo and in vitro assays and determined that GAPDH also interacts with Rab2. The site of GAPDH interaction was mapped to Rab2 residues 20-50. In addition to its glycolytic function, GAPDH has multiple intracellular roles. However, the function of GAPDH in the early secretory pathway is unknown. One possibility is that GAPDH ultimately provides energy in the form of ATP. To determine whether GAPDH catalytic activity was critical for transport in the early secretory pathway, a conservative substitution was made at Cys-149 located at the active site, and the mutant was biochemically characterized in a battery of assays. Although GAPDH (C149G) has no catalytic activity, Rab2 recruited the mutant protein to membranes in a quantitative binding assay. GAPDH (C149G) is phosphorylated by aPKC iota/lambda and binds directly to Rab2 when evaluated in an overlay binding assay. Importantly, VSV-G transport between the ER and Golgi complex is restored when an in vitro trafficking assay is performed with GAPDH-depleted cytosol and GAPDH (C149G). These data suggest that GAPDH imparts a unique function necessary for membrane trafficking from VTCs that does not require GAPDH glycolytic activity.

GAPDH enhances group II intron splicing in vitro.

Group II introns are autocatalytic RNAs which self-splice in vitro. However, in vivo additional protein factors might be involved in the splicing process. We used an affinity chromatography method called 'StreptoTag' to identify group II intron binding proteins from Saccharomyces cerevisiae. This method uses a hybrid RNA consisting of a streptomycin-binding affinity tag and the RNA of interest, which is bound to a streptomycin column and incubated with yeast protein extract. After several washing steps the bound RNPs are eluted by addition of streptomycin. The eluted RNPs are separated and the proteins identified by mass-spectrometric analysis. Using crude extract from yeast in combination with a substructure of the bl1 group II intron (domains IV-VI) we were able to identify four glycolytic enzymes; glucose-6-phosphate isomerase (GPI), 3-phosphoglycerate kinase (PGK), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and triosephosphate isomerase (TPI). From these proteins GAPDH increases in vitro splicing of the bl1 group II intron by up to three times. However, in vivo GAPDH is not a group II intron-splicing factor, since it is not localised in yeast mitochondria. Therefore, the observed activity reflects an unexpected property of GAPDH. Band shift experiments and UV cross linking demonstrated the interaction of GAPDH with the group II intron RNA. This novel activity expands the reaction repertoire of GAPDH to a new RNA species.

GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE ASSOCIATES WITH ACTIN FILAMENTS IN SERUM DEPRIVED NIH 3T3 CELLS ONLY

The in vitro interaction between the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and cytoskeletal elements is well documented. To verify this association within cells, the intracellular distribution of GAPDH under various metabolic conditions has been investigated in immunostained cells or cells expressing GAPDH as a GFP fusion protein. GAPDH was homogeneously distributed in the cytoplasm and no interaction of GAPDH with cytoskeletal elements, neither with microfilaments nor microtubules or intermediate filaments, was detectable. In living cells expressing GFP-GAPDH, stress fibres were excluded from the fluorescence. In contrast to proliferating cells, the cytoplasmic GAPDH of serum-depleted cells was not homogeneously distributed, but colocalised with stress fibres. The mechanism for stimulating this actin-binding affinity was independent of the NO-signalling pathway. The results support the idea of a specialised function for the interaction of GAPDH and cytoskeletal elements, rather than a general function, as e.g. microcompartmentalization of glycolytic enzymes.

Membrane-bound glyceraldehyde-3-phosphate dehydrogenase and multiphasic erythrocyte sugar transport.

Net sugar import by human erythrocytes consists of ATP-modulated rapid and slow phases while sugar export consists of a single slow phase. We have proposed that this behaviour results from obligate substrate tunnelling from transporter to bulk cytosol through a complex containing high-affinity, low-capacity sugar binding sites (Cloherty, Sultzman, Zottola & Carruthers, 1995). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is known to compartmentalize ATP delivery to erythrocyte membrane ATPases and interact directly with the erythrocyte glucose transporter in vitro. The present study examines the possibility that GAPDH is an obligate component of the hypothesized sugar-binding complex. GAPDH remains associated with the erythrocyte membrane following cell lysis and remains associated with the cytoskeleton under conditions where more than 99% of the membrane glucose transport protein (GLUT1) is released by detergent (Triton X-100). GAPDH is released from erythrocyte membranes upon exposure to Mg.ATP or to NADH. ATP displacement of membrane-bound GAPDH is half-maximal at 200 microM ATP and appears to involve ATP interaction with multiple, co-operative sites. GAPDH interaction with purified tetrameric GLUT1 is saturable, co-operative and also inhibited by ATP. ATP inhibition of GAPDH binding to purified tetrameric GLUT1 is less effective than ATP inhibition of GAPDH binding to intact erythrocyte membranes. Removal of cellular GAPDH by exposing erythrocyte membranes to NADH prior to membrane resealing neither affects ATP modulation of sugar transport nor reduces biphasic net sugar uptake to a single phase. We conclude that ATP-sensitive GAPDH interaction with the cytoplasmic surface of erythrocyte membranes and GLUT1 is responsible neither for ATP modulation of sugar transport nor for multiphasic net sugar import by human red cells.

Specific Interaction of Glyceraldehyde 3-Phosphate Dehydrogenase with the 5′-Nontranslated RNA of Hepatitis A Virus

Initiation of translation of hepatitis A virus (HAV) RNA occurs by internal entry and is likely to involve the interaction of trans-acting cellular protein factors with cis-acting structural elements of an internal ribosomal entry segment (IRES) within the 5'-nontranslated RNA. To characterize interactions between African green monkey kidney (BS-C-1) cell proteins and the predicted stem-loop IIIa (nucleotides 155-235) located at the 5' border of the HAV IRES, we utilized an electrophoresis mobility shift assay (EMSA) to identify a 39-kDa RNA-binding protein (p39). Amino-terminal amino acid sequencing of highly purified p39 revealed absolute identity with human glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The identity of p39 as simian GAPDH was further confirmed by antigenic and biochemical similarities between p39 and human GAPDH. Analysis of the RNA binding properties of simian GAPDH revealed that this cellular protein interacts with two additional sites in the HAV 5'-nontranslated RNA, one located between nucleotides 1-148 and the other between nucleotides 597-746. Competitive EMSAs also demonstrated that GAPDH and human polypyrimidine tract-binding protein, a putative picornavirus translation initiation factor, compete with each other for binding to stem-loop IIIa, suggesting that the relative cytoplasmic abundance of GAPDH and polypyrimidine tract-binding protein in individual cell-types may be an important determinant of viral translation activity. Human GAPDH was found to destabilize the folded structure of the stem-loop IIIa RNA based upon observed decreases in the circular dichroism spectra of this RNA following binding of the protein. This RNA helix-destabilizing activity of GAPDH could directly influence IRES-dependent translation and/or replication of picornavirus RNA.