Role of glyceraldehyde-3-phosphate dehydrogenase in vesicular transport from Golgi apparatus to endoplasmic reticulum

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a well-studied glycolytic protein with energy production as its implied occupation. It has established itself lately as a multifunctional protein. Recent studies have found GAPDH to be involved in a variety of nuclear and cytosolic pathways ranging from its role in apoptosis and regulation of gene expression to its involvement in regulation of Ca2+ influx from endoplasmic reticulum. Numerous studies also indicate that GAPDH interacts with microtubules and participates in cell membrane fusion. This review is focused on the cytosolic functions of the protein related to vesicular transport. Suggestions for future directions as well as the model of protein polymer structure and possible post-translational modifications as a basis for its multifunctional activities in the early secretory pathway are given.     

Glyceraldehyde-3-phosphate, a glycolytic intermediate, plays a key role in controlling cell fate via inhibition of caspase activity

Glyceraldehyde-3-phosphate is a key intermediate in several central metabolic pathways of all organisms. Aldolase and glyceraldehyde-3-phosphate dehydrogenase are involved in the production or elimination of glyceraldehyde-3-phosphate during glycolysis or gluconeogenesis, and are differentially expressed under various physiological conditions, including cancer, hypoxia, and apoptosis. In this study, we examine the effects of glyceraldehyde-3-phosphate on cell survival and apoptosis. Overexpression of aldolase protected cells against apoptosis, and addition of glyceraldehyde-3-phosphate to cells delayed apoptosis. Additionally, delayed apoptotic phenomena were observed when glyceraldehyde-3-phosphate was added to a cell-free system, in which artificial apoptotic process was induced by adding dATP and cytochrome c. Surprisingly, glyceraldehyde-3-phosphate directly suppressed caspase-3 activity in a reversible noncompetitive mode, preventing caspase-dependent proteolysis. Based on these results, we suggest that glyceraldehyde-3-phosphate, a key molecule in several central metabolic pathways, functions as a molecule switch between cell survival and apoptosis.     

Glyceraldehyde-3-phosphate dehydrogenase,a glycolytic enzyme present in the periplasm of Aeromonas hydrophila

This is the first report describing the glycolytic enzyme, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), as a protein associated with the cell envelope of a gram-negative bacterium (Aeromonas hydrophila). Dose-dependent GAPDH activity was detected in whole bacterial cells from exponentially growing cultures, indicating that an active form of GAPDH is located outside the plasma membrane. This activity represents roughly 10–20% of total cell activity, and it is not reduced by pretreatment of the cells with trypsin. Assays with soluble GAPDH indicate that the activity measured in intact cells does not originate by rebinding to intact cells of cytosolic enzyme released following cell lysis. GAPDH activity levels detected in intact cells varied during the growth phase. The relationship between GAPDH activity and cell culture density was not linear, showing this activity as a major peak in the late-logarithmic phase (A₆₀₀ = 1.1–1.3), and a decrease when cells entered the stationary phase. The late exponential growing cells showed a GAPDH activity 3 to 4-fold higher than early growing or stationary cells. No activity was detected in culture supernatants. Enzymatic and Western-immunoblotting analysis of subcellular fractions (cytosol, whole and outer membranes, and periplasm) showed that GAPDH is located in the cytosol, as expected, and also in the periplasm. These results place the periplasmic GAPDH of A. hydrophila into the family of multifunctional microbial cell wall-associated GAPDHs which retain their catalytic activity. This revised version was published online in June 2006 with corrections to the Cover Date.     

Glyceraldehyde-3-phosphate dehydrogenase activity as an independent modifier of methylglyoxal levels in diabetes

Methylglyoxal (MG) may be an important cause of diabetic complications. Its primary source is dihydroxyacetone phosphate (DHAP) whose levels are partially controlled by glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Using a human red blood cell (RBC) culture, we examined the effect of modifying GAPDH activity on MG production. With the inhibitor koningic acid (KA), we showed a linear, concentration-dependent GAPDH inhibition, with 5 microM KA leading to a 79% reduction of GAPDH activity and a sixfold increase in MG. Changes in redox state produced by elevated pH also resulted in a 2.4-fold increase in MG production at pH 7.5 and a 13.4-fold increase at pH 7.8. We found substantial inter-individual variation in DHAP and MG levels and an inverse relationship between GAPDH activity and MG production (R=0.57, P=0.005) in type 2 diabetes. A similar relationship between GAPDH activity and MG was observed in vivo in type 1 diabetes (R=0.29, P=0.0018).Widely varying rates of progression of diabetic complications are seen among individuals. We postulate that modification of GAPDH by environmental factors or genetic dysregulation and the resultant differences in MG production could at least partially account for this observation.     

Rab1 and Ca2+ are required for the fusion of carrier vesicles mediating endoplasmic reticulum to Golgi transport

Members of the rab/YPT1/SEC4 gene family of small molecular weight GTPases play key roles in the regulation of vesicular traffic between compartments of the exocytic pathway. Using immunoelectron microscopy, we demonstrate that a dominant negative rab1a mutant, rab1a(N124I), defective for guanine nucleotide binding in vitro, leads to the accumulation of vesicular stomatitis virus glycoprotein (VSV-G) in numerous pre-cis-Golgi vesicles and vesicular-tubular clusters containing rab1 and beta-COP, a subunit of the coatomer complex. Similar to previous observations (Balch et al. 1994. Cell. 76:841-852), VSV-G was concentrated nearly 5-10-fold in vesicular carriers that accumulate in the presence of the rab1a(N124I) mutant. VSV-G containing vesicles and vesicular-tubular clusters were also found to accumulate in the presence of a rab1a effector domain peptide mimetic that inhibits endoplasmic reticulum to Golgi transport, as well as in the absence of Ca2+. These results suggest that the combined action of a Ca(2+)-dependent protein and conformational changes associated with the GTPase cycle of rab1 are essential for a late targeting/fusion step controlling the delivery of vesicles to Golgi compartments.     

Guanine nucleotide dissociation inhibitor is essential for Rab1 function in budding from the endoplasmic reticulum and transport through the Golgi stack

The small GTPase Rab1 is required for vesicular traffic from the ER to the cis-Golgi compartment, and for transport between the cis and medial compartments of the Golgi stack. In the present study, we examine the role of guanine nucleotide dissociation inhibitor (GDI) in regulating the function of Rab1 in the transport of vesicular stomatitis virus glycoprotein (VSV-G) in vitro. Incubation in the presence of excess GDI rapidly (t1/2 < 30 s) extracted Rab1 from membranes, inhibiting vesicle budding from the ER and sequential transport between the cis-, medial-, and trans-Golgi cisternae. These results demonstrate a direct role for GDI in the recycling of Rab proteins. Analysis of rat liver cytosol by gel filtration revealed that a major pool of Rab1 fractionates with a molecular mass of approximately 80 kD in the form of a GDI-Rab1 complex. When the GDI-Rab1 complex was depleted from cytosol by use of a Rab1-specific antibody, VSV-G failed to exit the ER. However, supplementation of depleted cytosol with a GDI-Rab1 complex prepared in vitro from recombinant forms of Rab1 and GDI efficiently restored export from the ER, and transport through the Golgi stack. These results provide evidence that a cytosolic GDI-Rab1 complex is required for the formation of non-clathrin-coated vesicles mediating transport through the secretory pathway.     

Beta-COP is essential for transport of protein from the endoplasmic reticulum to the Golgi in vitro

Using a novel in vitro assay which allows us to distinguish vesicle budding from subsequent targeting and fusion steps, we provide the first biological evidence that beta-COP, a component of non-clathrin- coated vesicles believed to mediate intraGolgi transport, is essential for transport of protein from the ER to the cis-Golgi compartment. Incubation in the presence of beta-COP specific antibodies and F(ab) fragments prevents the exit of vesicular stomatitis virus glycoprotein (VSV-G) from the ER. These results demonstrate that beta-COP is required for the assembly of coat complexes mediating vesicle budding. Fractionation of rat liver cytosol revealed that a major biologically active form of beta-COP was found in a high molecular pool (> 1,000 kD) distinct from coatomer and which promoted efficient vesicle budding from the ER. Surprisingly, rab1B could be quantitatively coprecipitated with this beta-COP containing complex and was also essential for function. We suggest that beta-COP functions in an early step during vesicle formation and that rab1B may be recruited as a component of a precoat complex which participates in the export of protein from the ER via vesicular carriers.     

Glyceraldehyde-3-phosphate dehydrogenase interacts with phosphorylated Akt resulting from increased blood glucose in rat cardiac muscle

Here we describe the interaction of phosphorylated ∼40 kDa protein with phosphorylated Akt which is a serine/threonine kinase resulting from increased blood glucose in rat cardiac muscle. Mass spectrometry analysis revealed that this protein was glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Furthermore, increase in Akt and GAPDH phosporylation and induction of their association were both observed after insulin stimulation in the H9c2 cell line derived from embryonic rat ventricle. Moreover, the activation of GAPDH was upregulated when the GAPDH phosphorylation was increased. Our data suggest that GAPDH phosphorylation and association with Akt by insulin treatment have some bearing on the enhancement of GAPDH activity.

Structured summary

MINT-7891324, MINT-7891304, MINT-7891314: GAPDH (uniprotkb:P04797) physically interacts (MI:0915) with Akt (uniprotkb:P47196) by anti bait coimmunoprecipitation (MI:0006)     

Organization of transport from endoplasmic reticulum to Golgi in higher plants

In plant cells, the organization of the Golgi apparatus and its interrelationships with the endoplasmic reticulum differ from those in mammalian and yeast cells. Endoplasmic reticulum and Golgi apparatus can now be visualized in plant cells in vivo with green fluorescent protein (GFP) specifically directed to these compartments. This makes it possible to study the dynamics of the membrane transport between these two organelles in the living cells. The GFP approach, in conjunction with a considerable volume of data about proteins participating in the transport between endoplasmic reticulum and Golgi in yeast and mammalian cells and the identification of their putative plant homologues, should allow the establishment of an experimental model in which to test the involvement of the candidate proteins in plants. As a first step towards the development of such a system, we are using Sar1, a small G-protein necessary for vesicle budding from the endoplasmic reticulum. This work has demonstrated that the introduction of Sar1 mutants blocks the transport from endoplasmic reticulum to Golgi in vivo in tobacco leaf epidermal cells and has therefore confirmed the feasibility of this approach to test the function of other proteins that are presumably involved in this step of endomembrane trafficking in plant cells.     

Identification of an intermediate compartment involved in protein transport from endoplasmic reticulum to Golgi apparatus

We have studied the role of a previously described tubulovesicular compartment near the cis-Golgi apparatus in endoplasmic reticulum (ER)-to-Golgi protein transport by light and immunoelectron microscopy in Vero cells. The compartment is defined by a 53-kDa transmembrane protein designated p53. When transport of the vesicular stomatitis virus strain ts045 G protein was arrested at 39.5 degrees C, the G protein accumulated in the ER but had access to the p53 compartment. At 15 degrees C, the G protein was exported from the ER into the p53 compartment which formed a compact structure composed of vesicular and tubular profiles in close proximity to the Golgi. Upon raising the temperature to 32 degrees C, the G protein migrated through the Golgi apparatus while the p53 compartment resumed its normal structure again. These results establish the p53 compartment as the 15 degrees C intermediate of the ER-to-Golgi protein transport pathway.     

A GFP-based assay reveals a role for RHD3 in transport between the endoplasmic reticulum and Golgi apparatus

We describe the use of a secreted form of Aequoria victoria green fluorescent protein (secGFP) in a non-invasive live cell assay of membrane traffic in Arabidopsis thaliana. We show that in comparison to GFP-HDEL, which accumulates in the endoplasmic reticulum (ER), secGFP generates a weak fluorescence signal when transported to the apoplast. The fluorescence of secGFP in the apoplast can be increased by growth of seedlings on culture medium buffered at pH 8.1, suggesting that apoplastic pH is responsible, at least in part, for the low fluorescence intensity of seedlings expressing secGFP. Inhibition of secGFP transport between the ER and plasma membrane (PM), either by Brefeldin A (BFA) treatment or by genetic intervention results in increased intracellular secGFP accumulation accompanied by an increase in the secGFP fluorescence intensity. secGFP thus provides a valuable tool for forward and reverse genetic analysis of membrane traffic and endomembrane organisation in Arabidopsis. Using this assay for quantitative sublethal perturbation of secGFP transport, we identify a role for root hair defective 3 (RHD3) in transport of secreted and Golgi markers between the ER and the Golgi apparatus.     

The role of microtubules in transport between the endoplasmic reticulum and Golgi apparatus in mammalian cells

The organization of intracellular compartments and the transfer of components between them are central to the correct functioning of mammalian cells. Proteins and lipids are transferred between compartments by the formation, movement and subsequent specific fusion of transport intermediates. These vesicles and membrane clusters must be coupled to the cytoskeleton and to motor proteins that drive motility. Anterograde ER (endoplasmic reticulum)-to-Golgi transport, and the converse step of retrograde traffic from the Golgi to the ER, are now known to involve coupling of membranes to the microtubule cytoskeleton. Here we shall discuss our current understanding of the mechanisms that link membrane traffic in the early secretory pathway to the microtubule cytoskeleton in mammalian cells. Recent data have also provided molecular detail of functional co-ordination of motor proteins to specify directionality, as well as mechanisms for regulating motor activity by protein phosphorylation.     

Sequential coupling between COPII and COPI vesicle coats in endoplasmic reticulum to Golgi transport

COPI and COPII are vesicle coat complexes whose assembly is regulated by the ARF1 and Sar1 GTPases, respectively. We show that COPI and COPII coat complexes are recruited separately and independently to ER (COPII), pre-Golgi (COPI, COPII), and Golgi (COPI) membranes of mammalian cells. To address their individual roles in ER to Golgi transport, we used stage specific in vitro transport assays to synchronize movement of cargo to and from pre-Golgi intermediates, and GDP- and GTP-restricted forms of Sar1 and ARF1 proteins to control coat recruitment. We find that COPII is solely responsible for export from the ER, is lost rapidly following vesicle budding and mediates a vesicular step required for the build-up of pre-Golgi intermediates composed of clusters of vesicles and small tubular elements. COPI is recruited onto pre-Golgi intermediates where it initiates segregation of the anterograde transported protein vesicular stomatitis virus glycoprotein (VSV-G) from the retrograde transported protein p58, a protein which actively recycles between the ER and pre-Golgi intermediates. We propose that sequential coupling between COPII and COPI coats is essential to coordinate and direct bi-directional vesicular traffic between the ER and pre-Golgi intermediates involved in transport of protein to the Golgi complex.     

Glyceraldehyde-3-phosphate dehydrogenase is phosphorylated by protein kinase Ciota /lambda and plays a role in microtubule dynamics in the early secretory pathway.

The small GTPase Rab2 immunolocalizes to vesicular tubular clusters (VTCs) that function as transport complexes carrying cargo between the endoplasmic reticulum and the Golgi complex. Our previous studies showed that Rab2 promotes vesicle formation from VTCs and that the released vesicles are enriched in beta-coat protein, protein kinase C iota/lambda (PKCiota/lambda), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and the recycling protein p53/gp58. Because PKCiota/lambda kinase activity was necessary for vesicle formation, a search was initiated to identify the substrate(s) that potentiate Rab2 function within VTCs. In this study, we found that PKCiota/lambda phosphorylates GAPDH. Moreover, GAPDH interacts directly with the PKCiota/lambda regulatory domain. Based on numerous observations that show (beta-COP) GAPDH associates with cytoskeletal elements, we examined the role of phospho-GAPDH in promoting microtubule (MT) binding to membrane. Using a quantitative microsomal binding assay, we found that membrane association of beta-tubulin was dependent on phospho-GAPDH and was blocked by reagents that interfere with Rab2-dependent GAPDH membrane recruitment or with PKCiota/lambda kinase activity. Furthermore, normal rat kidney cells transfected with a constitutively activated form of Rab2 (Q65L) or with our anti-GAPDH polyclonal antibody displayed a dramatic change in MT organization. These combined results suggest that Rab2 stimulated PKCiota/lambda and GAPDH recruitment to VTCs, and the subsequent PKCiota/lambda phosphorylation of GAPDH ultimately influences MT dynamics in the early secretory pathway.     

Glyceraldehyde-3-phosphate dehydrogenase exerts different biologic activities in apoptotic and proliferating hepatocytes according to its subcellular localization

Recent evidences indicate new roles for the glycolytic protein glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in essential mammalian cell processes, such as apoptosis and proliferation. To clarify the involvement of this protein in growth and programmed cell death in the liver, cell models of hepatocytes in culture were used to study GAPDH expression, localization and enzymatic activity in hepatocyte proliferation and apoptosis. GAPDH expression in cell compartments was studied by Western blot. Nuclear expression of GAPDH increased in apoptosis, and cytoplasmic expression was elevated in apoptosis and proliferation. Subcellular localization was determined by GAPDH immunostaining and confocal microscopic analysis. Quiescent and proliferating hepatocytes showed cytoplasmic GAPDH, while apoptotic cells showed cytoplasmic but also some nuclear staining. The glycolytic activity of GAPDH was studied in nuclear and cytoplasmic cell compartments. GAPDH enzymatic activity increased in the nucleus of apoptotic cells and in cytoplasms of apoptotic and proliferating hepatocytes. Our observations indicate that during hepatocyte apoptosis GAPDH translocates to the nucleus, maintaining in part its dehydrogenase activity, and suggest that this translocation may play a role in programmed hepatocyte death. GAPDH over-expression and the increased enzymatic activity in proliferating cells, with preservation of its cytoplasmic localization, would occur in response to the elevated energy requirements of dividing hepatocytes. In conclusion, GAPDH plays different roles or biological activities in proliferating and apoptotic hepatocytes, according to its subcellular localization.     

Glyceraldehyde-3-phosphate dehydrogenase from Ehrlich ascites carcinoma cells its possible role in the high glycolysis of malignant cells.

Glyceraldehyde-3-phosphate dehydrogenase has been purified to apparent homogeneity from Ehrlich ascites carcinoma (EAC) cells. The enzyme is quite active over a pH range of 7.5-9.0 with an optimum pH of 8.4-8.7. The specific activity of the enzyme is much higher than that from other normal sources. In contrast to enzyme obtained from rabbit muscle, the EAC cell enzyme is not significantly inhibited by physiological concentrations of ATP at physiological pH. Kinetic studies using different substrates and inhibitors indicate that the properties of the EAC cell enzyme are significantly different from those of glyceraldehyde-3-phosphate dehydrogenase obtained from other normal sources. The striking dissimilarity of the malignant cell glyceraldehyde-3-phosphate dehydrogenase compared with this enzyme from other normal sources, particularly in respect to the interaction with ATP, may in part explain the high glycolysis of malignant cells.     

Rab2 GTPase regulates vesicle trafficking between the endoplasmic reticulum and the Golgi bodies and is important to pollen tube growth

Pollen tube elongation depends on the secretion of large amounts of membrane and cell wall materials at the pollen tube tip to sustain rapid growth. A large family of RAS-related small GTPases, Rabs or Ypts, is known to regulate both anterograde and retrograde trafficking of transport vesicles between different endomembrane compartments and the plasma membrane in mammalian and yeast cells. Studies on the functional roles of analogous plant proteins are emerging. We report here that a tobacco pollen-predominant Rab2, NtRab2, functions in the secretory pathway between the endoplasmic reticulum and the Golgi in elongating pollen tubes. Green fluorescent protein-NtRab2 fusion protein localized to the Golgi bodies in elongating pollen tubes. Dominant-negative mutations in NtRab2 proteins inhibited their Golgi localization, blocked the delivery of Golgi-resident as well as plasmalemma and secreted proteins to their normal locations, and inhibited pollen tube growth. On the other hand, when green fluorescent protein-NtRab2 was over-expressed in transiently transformed leaf protoplasts and epidermal cells, in which NtRab2 mRNA have not been observed to accumulate to detectable levels, these proteins did not target efficiently to Golgi bodies. Together, these observations indicate that NtRab2 is important for trafficking between the endoplasmic reticulum and the Golgi bodies in pollen tubes and may be specialized to optimally support the high secretory demands in these tip growth cells.