Glycolysis involves the conversion of glucose to pyruvate with energy generation in the form of ATP . This metabolic pathway is achieved using ten enzymes (Fig. 1):
Being the basis of both aerobic and anaerobic respiration, glycolysis is one of the most fundamental metabolic pathways. In fact, it is present in most known organisms . Understanding the organization and regulation of glycolysis is therefore vital to understanding metabolism. There is now significant evidence that glycolytic enzymes associate as a metabolon. This has been well-studied in mammalian muscle cells, erythrocytes, and bacteria [3,4]. However, most of this evidence comes from in vitro work, including kinetic studies and characterization of protein-protein interactions. Recent reviews have highlighted a need for in vivo study and validation of this putative metabolon .
Of all model organisms, the glycolytic metabolon of S. cerevisiae is surprisingly under-studied. Recent work by Araiza-Olivera et al. provided the first strong evidence for a glycolytic metabolon in yeast . The glycolytic activity of yeast cells permeabilized with streptolysin O was found to be resistant to inhibition by targeted antibodies or viscogens. In contrast, the isolated glycolytic enzymes were shown to be susceptible to inhibition by these agents. This kinetic protection is a typical feature of metabolons. Furthermore, it is proposed that this metabolon interacts directly with and is stabilized by filamentous F-actin. In support of this, ALD and GAPD were shown to bind yeast F-actin in co-sedimentation experiments . More recently, co-immunoprecipitation experiments indicated that all but three (PFK, PGM and PK) of the ten yeast glycolytic enzymes bind F-actin . In contrast, none of them co-immunoprecipitated with monomeric G-actin. Lastly, disruption of F-actin by cytochalasin D reduced the rate of glycolysis supported by a yeast cytoplasmic extract . This data makes a strong case for the existence of a glycolytic metabolon in S. cerevisiae. However, several deficiencies in the current body of evidence are apparent. Firstly, evidence for the expected protein-protein interactions between sequential enzymes of the pathway is currently lacking. Secondly, despite compelling indirect evidence, it has not yet been explicitly shown that F-actin stabilizes the metabolon’s protein-protein interactions. Lastly, there is currently no in vivo evidence of a glycolytic metabolon in S. cerevisiae, all studies to date have used in vitro methods. To address these limitations, the proposed research aims to characterize the protein-protein interactions between the enzymes of the glycolytic metabolon of S. cerevisiae in vitro and in vivo. Furthermore, the contribution of F-actin to the stability of this complex will be elucidated. Based on the current literature evidence, a model for the S. cerevisiae glycolytic metabolon complex is proposed in Fig. 2.
Proposal – Experiments
Interactions between glycolytic enzymes in vitro
This FRET-based approach has several advantages over other techniques available to characterize protein-protein interactions. Unlike many techniques, it is suitable for characterization both in vitro and in vivo . Furthermore, the FRET interaction is reversible and highly dependent on distance, making false positives unlikely. In contrast, bimolecular fluorescence complementation is more sensitive than FRET, but the interaction is irreversible . This makes false positives a problem and limits the types of experiments that can be done. The yeast-2-hybrid assay is another approach to study protein-protein interactions in vivo . However, this assay reports on interactions in the nucleus, while the interactions of interest occur in the cytoplasm and may require F-actin for stability. Furthermore, metabolon protein-protein interactions are generally of relatively low affinity . Therefore, tandem affinity purification, which requires robust interactions, is also not a viable option.
Several control experiments are required to validate the FRET-based approach. As negative controls, the interaction of CFP and YFP alone will be tested in the above experiment. This will verify that interactions observed are not due to non-specific interactions between the fluorescent proteins. As an additional negative control, the interaction between glycolytic enzymes and Hog1 will be studied. Hog1 is known not to interact with yeast glycolytic enzyme . We expect interactions between all enzymes which are consecutive in the metabolic pathway. A direct physical interaction is essential to allow substrate channeling. If any of these interactions are not detected by this assay, there are several alternative explanations. Firstly, F-actin may be essential to stabilize the interaction. If this is the case, it will be clarified when the experiment is repeated in the presence of F-actin, as described below. The interaction may also fail to be detected due to the design of the fusion proteins. The fluorescent protein tags may interfere with the proper folding of the enzymes, or may block the interaction face. This problem may be resolved by switching the position of the fluorescent protein tag on either protein (N or C-terminus), or by adjusting the linker length.
F-actin binding of glycolytic enzymes in vitro
To ensure that changes in the FRAP recovery rates are the consequence of specific interactions between the glycolytic enzymes and F-actin, CFP and YFP will be used as negative controls. To further verify the specificity, the previously mentioned Hog1-fluoresent protein tagged construct can also be used as a negative control. All glycolytic enzymes are expected to interact with F-actin, save PFK, PGM, and PK. If any of the expected interactions are not observed, we must again be concerned about the effects of the fusion construction. The fluorescent protein tags may interfere with the proper folding of the enzymes, or may block the interaction face with F-actin. The problem may be resolved by switching the position of the fluorescent protein tag on either protein (N or C-terminus), or by adjusting the linker length.
The effect of F-actin on glycolytic enzymes interactions in vitro
Glycolytic enzyme protein-protein interactions in vivo
Next, the role of F-actin in the stability of the glycolytic complex can be probed in vivo using the validated protein-protein interactions. After observing an in vivo interaction by FRET, the cells can be treated with cytochalasin D. This compound inhibits actin polymerization, resulting in the rapid disintegration of F-actin filaments . If F-actin is essential for the metabolon’s stability, we expect dissociation of the complex and abolition of the FRET signal (Fig. 4). After a sufficient washout period to allow clearance of the drug, the FRET signal is expected to return concomitantly with the reformation of F-actin and the metabolon complex. This process can also be monitored by FRAP. Yeast cells modified with one fluorescent protein-tagged glycolytic enzyme will be used. The tagged enzyme is expected to have an attenuated diffusion rate and fluorescence recovery while bound in a metabolon complex. As before, the cells can then be treated with cytochalasin D to disrupt the F-actin polymers. Given that this reduces the stability of the metabolon complex and causes it to dissociate, an increase in the diffusion rate and fluorescence recovery rate of the tagged enzyme is expected.
As with the in vitro experiments, negative controls are essential. Yeast cells will be transformed with a plasmid encoding YFP and CFP and used as a negative control. This will ensure that YFP and CFP do not associate non-specifically in the cellular environment, and that the effect of cytochalasin D in the FRAP experiment is specific. Additionally, the Hog1 yeast chromosome gene will also be tagged with a fluorescent protein and tested for interaction with the glycolytic enzymes as a negative control in the in vivo FRET experiments. A possible limitation of using FRET in vivo is its sensitivity. Detecting interactions in vivo with dissociation constants greater than 10 μM is likely unfeasible . Thus, it will be most efficient to focus in vivo work on protein-protein interactions found to have sufficient affinity in vitro. A few in vivo interactions will be enough to show the existence of the metabolon and elucidate the role of F-actin. It would be possible to detect low affinity interactions in vivo with a more sensitive technique, like bimolecular fluorescence complementation . However, this technique is undesirable due to its irreversibility, which would preclude many of the above experiments.
An additional consequence of this research will be the development of new tools to study yeast glycolysis in vivo. If successful, the FRET-based approach will provide a versatile in vivo probe for the status of the glycolytic metabolon. It has been proposed that the dynamic association/dissociation of metabolon complexes could be a means to regulate metabolic flux . The technique will permit observation of these dynamic processes in living cells, allowing this hypothesis to be experimentally tested. Given the putative role of F-actin in the glycolytic complex’s stability, it is interesting that yeast F-actin structure and its complement of bound proteins is known to be highly regulated and dynamic . An intriguing hypothesis emerges from this, that F-actin binding could be a biologically relevant means of glycolytic regulation. In the future, this FRET-based technique may allow detailed in vivo experiments correlating glycolytic metabolon association, glycolysis rate, and metabolic state.
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