Some unanswered questions
| General | What are the amounts and stoichiometries of the membrane proteins (only a few are accurately measured)? |
| Spectrin | What is the structure of spectrin in vivo? How does it change when the membrane is deformed? |
| How does spectrin bind to the actin protofilament? What is the stoichiometry? Where do the CH1 and CH2 domains and protein 4.1R bind on actin? Is there >1 binding conformation? If so, how do these differ functionally and how are they regulated? | |
| Why is the EF hand domain essential for normal spectrin-actin binding? How does it work? | |
| How do spectrin molecules, which lie in a plane, accommodate different orientations of their binding sites on the helical actin protofilament? | |
| Higher order spectrin oligomers (hexamers, octamers, etc) also serve as molecular junctions in the skeleton. Are there significant numbers of these and do they have a unique functional role? | |
| If the available spectrin repeat structures are correct and there is a continuous α-helix across the interrepeat junction, how is spectrin flexibility achieved? | |
| Why are the same repeats in different spectrins so similar? Do they have binding or mechanical functions that we do not yet know about? | |
| What binds to the spectrin src homology 3 (SH3) domain? What are the consequences? | |
| There is evidence that some of the spectrins in mature red cells contain the muscle isoform of β1-spectrin, which has a lipid-interacting pleckstrin homology domain. Is this true, and if so, what is its function? | |
| Actin | How are actin protofilaments formed? How do they achieve their uniform size? Do formins play a role? |
| Are the actin protofilaments stable or do they turn over, as the presence of a critical concentration of G-actin in the red cell cytoplasm suggests? If so, how is this regulated? | |
| How are the various proteins arranged on the actin protofilament? Is the arrangement the same in all the protofilaments? If there are open binding sites on actin, do molecules like spectrin move from site to site with membrane deformation? | |
| Do red cell myosin and caldesmon have a function in the membrane of mature red cells? | |
| Ankyrin | Does ankyrin bind a band 3 tetramer or 2 dimers? If a tetramer, is it preformed before binding or do 2 dimers bind and convert to a tetramer on ankyrin. What is the structure of the band 3 tetramer-ankyrin complex? |
| What is the structure of the ankyrin molecule? How do the various domains relate to each other spatially? | |
| Where do protein 4.2 and RhAG bind? | |
| What is the function of the death domain in ankyrin? | |
| What are the functions of the many ankyrin spliceoforms? | |
| Band 3 | What is the overall structure of band 3? |
| Where do protein 4.2 and adducin bind on band 3? | |
| Because the numbers of integral membrane proteins vary by orders of magnitude (Table 1), the band 3 multiprotein complexes must vary in composition. How many such complexes are there? Do they have specific, stable compositions or are they in equilibrium and continually changing their composition? Are the substoichiometric proteins (eg, CD44, CD47, LW, Kx/Kell, DARC) uniformly distributed or localized to lipid rafts or other membrane lipid or protein subdomains? | |
| How are the proteins in the band 3 multiprotein complexes arranged relative to each other? | |
| Do protein interactions within the band 3 multiprotein complexes allosterically affect the interactions or functions of other proteins in the complexes? | |
| Does protein 4.1R bind to band 3 in vivo? If so, does band 3 bind to each of the spectrin/actin/protein 4.1R complexes or only to a subset, such as the subset that interacts with adducin (Figure 8)? | |
| Are any band 3 molecules “unbound” (ie, not attached to the membrane skeleton directly or indirectly). If so, how many? Are they also part of multiprotein complexes? Do they have a unique function? | |
| Glycolytic metabolon | The enzymes that compose the proposed glycolytic metabolon vary in concentration by orders of magnitude. Are the estimates correct? If so, do some complexes lack rare components (but then, how would they function?) or are they gigantic so as to include at least 1 copy of each enzyme? Are other enzymes in the glycolytic pathway part of the complex? |
| How is the glycolytic metabolon regulated in vivo? What is the purpose of activating glycolysis by deoxyhemoglobin? Do products of glycolysis such as adenosine triphosphate (ATP) cause vasodilation directly or indirectly? Does nitric oxide play a role? Is the rate of dissociation and activation of the glycolytic metabolon rapid enough to be relevant during normal circulatory transits? | |
| Protein 4.1R | What is the structure of intact protein 4.1R and the spectrin/actin/protein 4.1R complex? |
| Calmodulin binds to and regulates protein 4.1R. The EF hand domain is a calmodulin-like structure that resides near protein 4.1R in the actin junctional complex. In addition, the EF domain binds calmodulin. Does protein 4.1R bind to the EF domain or do they bind each other through calmodulin? If so, is this a regulatory interaction? | |
| Does phosphatidyl-4,5-bisphosphate regulate protein 4.1R binding to actin or other proteins in vivo? | |
| Because there is insufficient protein p55/palmitoylated membrane protein to make a 1:1:1 molar complex with protein 4.1R and glycophorin C/D, how is the complex constructed? | |
| Protein 4.2 | What is the function of protein 4.2 when attached to the spectrin EF hand domain? Does it actually occupy the domain in vivo? If so, does it bind to protein 4.1R? |
| What is the function of the ATP-binding site on protein 4.2? | |
| Adducin | Is adducin a dimer or a tetramer? |
| What is the structure of adducin? How does the C-terminal tail relate to the head end? | |
| Band 3, spectrin, and actin all bind to the C-terminal tail. What is the structure of this complex? How are the multiple interactions regulated? Can they occur all at once on the same molecule or do some interactions interfere with others? When adducin binds spectrin and actin, does it also interact with nearby proteins 4.1R or 4.2? Does adducin bind band 3 dimers or tetramers, and does it bind 1 or 2 molecules of each? | |
| Dematin | How does dematin contribute to spectrin binding in vivo? |
| Is the ability of dematin to bundle actin filaments important in the mature red cell? | |
| Where does dematin bind relative to spectrin and adducin on the actin protofilament? Does the number of dematins per protofilament vary? | |
| Tropomyosin | Do the red cell isoforms of tropomyosin determine the unique structure of the short actin protofilaments? Do specific actin-nucleating formins play a role in this process? |
| Posttranslational modifications | What do posttranslational modifications like phosphorylation, palmitoylation, myristoylation, hydroxylation, methylation, glycation, and ubiquitination do? Are the effects important in vivo? |
| How are the multiple effects of calcium ion and calmodulin integrated in vivo? | |
| Dynamics | Do the actin junctional complex and ankyrin complex contact each other in vivo? Do proteins like band 3, proteins 4.1R and 4.2, and adducin, which have potential binding partners in both complexes, sometimes switch allegiances? If so, is this a regulatory process? |
| How labile are the individual bonds in the membrane skeleton? Which bonds, other than the spectrin dimer-tetramer interaction, break when the skeleton is deformed? Are the rates of dissociation and reassociation such that the bonds dissociate during red cell deformation in the circulation? | |
| How is the skeleton disassembled and reassembled during invasion by malaria and other parasites? | |
| Lipids | What are the interactions between the membrane skeleton and the overlying lipids? Do these interactions have regulatory as well as structural functions? Does the membrane skeleton vary in unique regions of the lipid bilayer such as lipid rafts? |
| General | What are the amounts and stoichiometries of the membrane proteins (only a few are accurately measured)? |
| Spectrin | What is the structure of spectrin in vivo? How does it change when the membrane is deformed? |
| How does spectrin bind to the actin protofilament? What is the stoichiometry? Where do the CH1 and CH2 domains and protein 4.1R bind on actin? Is there >1 binding conformation? If so, how do these differ functionally and how are they regulated? | |
| Why is the EF hand domain essential for normal spectrin-actin binding? How does it work? | |
| How do spectrin molecules, which lie in a plane, accommodate different orientations of their binding sites on the helical actin protofilament? | |
| Higher order spectrin oligomers (hexamers, octamers, etc) also serve as molecular junctions in the skeleton. Are there significant numbers of these and do they have a unique functional role? | |
| If the available spectrin repeat structures are correct and there is a continuous α-helix across the interrepeat junction, how is spectrin flexibility achieved? | |
| Why are the same repeats in different spectrins so similar? Do they have binding or mechanical functions that we do not yet know about? | |
| What binds to the spectrin src homology 3 (SH3) domain? What are the consequences? | |
| There is evidence that some of the spectrins in mature red cells contain the muscle isoform of β1-spectrin, which has a lipid-interacting pleckstrin homology domain. Is this true, and if so, what is its function? | |
| Actin | How are actin protofilaments formed? How do they achieve their uniform size? Do formins play a role? |
| Are the actin protofilaments stable or do they turn over, as the presence of a critical concentration of G-actin in the red cell cytoplasm suggests? If so, how is this regulated? | |
| How are the various proteins arranged on the actin protofilament? Is the arrangement the same in all the protofilaments? If there are open binding sites on actin, do molecules like spectrin move from site to site with membrane deformation? | |
| Do red cell myosin and caldesmon have a function in the membrane of mature red cells? | |
| Ankyrin | Does ankyrin bind a band 3 tetramer or 2 dimers? If a tetramer, is it preformed before binding or do 2 dimers bind and convert to a tetramer on ankyrin. What is the structure of the band 3 tetramer-ankyrin complex? |
| What is the structure of the ankyrin molecule? How do the various domains relate to each other spatially? | |
| Where do protein 4.2 and RhAG bind? | |
| What is the function of the death domain in ankyrin? | |
| What are the functions of the many ankyrin spliceoforms? | |
| Band 3 | What is the overall structure of band 3? |
| Where do protein 4.2 and adducin bind on band 3? | |
| Because the numbers of integral membrane proteins vary by orders of magnitude (Table 1), the band 3 multiprotein complexes must vary in composition. How many such complexes are there? Do they have specific, stable compositions or are they in equilibrium and continually changing their composition? Are the substoichiometric proteins (eg, CD44, CD47, LW, Kx/Kell, DARC) uniformly distributed or localized to lipid rafts or other membrane lipid or protein subdomains? | |
| How are the proteins in the band 3 multiprotein complexes arranged relative to each other? | |
| Do protein interactions within the band 3 multiprotein complexes allosterically affect the interactions or functions of other proteins in the complexes? | |
| Does protein 4.1R bind to band 3 in vivo? If so, does band 3 bind to each of the spectrin/actin/protein 4.1R complexes or only to a subset, such as the subset that interacts with adducin (Figure 8)? | |
| Are any band 3 molecules “unbound” (ie, not attached to the membrane skeleton directly or indirectly). If so, how many? Are they also part of multiprotein complexes? Do they have a unique function? | |
| Glycolytic metabolon | The enzymes that compose the proposed glycolytic metabolon vary in concentration by orders of magnitude. Are the estimates correct? If so, do some complexes lack rare components (but then, how would they function?) or are they gigantic so as to include at least 1 copy of each enzyme? Are other enzymes in the glycolytic pathway part of the complex? |
| How is the glycolytic metabolon regulated in vivo? What is the purpose of activating glycolysis by deoxyhemoglobin? Do products of glycolysis such as adenosine triphosphate (ATP) cause vasodilation directly or indirectly? Does nitric oxide play a role? Is the rate of dissociation and activation of the glycolytic metabolon rapid enough to be relevant during normal circulatory transits? | |
| Protein 4.1R | What is the structure of intact protein 4.1R and the spectrin/actin/protein 4.1R complex? |
| Calmodulin binds to and regulates protein 4.1R. The EF hand domain is a calmodulin-like structure that resides near protein 4.1R in the actin junctional complex. In addition, the EF domain binds calmodulin. Does protein 4.1R bind to the EF domain or do they bind each other through calmodulin? If so, is this a regulatory interaction? | |
| Does phosphatidyl-4,5-bisphosphate regulate protein 4.1R binding to actin or other proteins in vivo? | |
| Because there is insufficient protein p55/palmitoylated membrane protein to make a 1:1:1 molar complex with protein 4.1R and glycophorin C/D, how is the complex constructed? | |
| Protein 4.2 | What is the function of protein 4.2 when attached to the spectrin EF hand domain? Does it actually occupy the domain in vivo? If so, does it bind to protein 4.1R? |
| What is the function of the ATP-binding site on protein 4.2? | |
| Adducin | Is adducin a dimer or a tetramer? |
| What is the structure of adducin? How does the C-terminal tail relate to the head end? | |
| Band 3, spectrin, and actin all bind to the C-terminal tail. What is the structure of this complex? How are the multiple interactions regulated? Can they occur all at once on the same molecule or do some interactions interfere with others? When adducin binds spectrin and actin, does it also interact with nearby proteins 4.1R or 4.2? Does adducin bind band 3 dimers or tetramers, and does it bind 1 or 2 molecules of each? | |
| Dematin | How does dematin contribute to spectrin binding in vivo? |
| Is the ability of dematin to bundle actin filaments important in the mature red cell? | |
| Where does dematin bind relative to spectrin and adducin on the actin protofilament? Does the number of dematins per protofilament vary? | |
| Tropomyosin | Do the red cell isoforms of tropomyosin determine the unique structure of the short actin protofilaments? Do specific actin-nucleating formins play a role in this process? |
| Posttranslational modifications | What do posttranslational modifications like phosphorylation, palmitoylation, myristoylation, hydroxylation, methylation, glycation, and ubiquitination do? Are the effects important in vivo? |
| How are the multiple effects of calcium ion and calmodulin integrated in vivo? | |
| Dynamics | Do the actin junctional complex and ankyrin complex contact each other in vivo? Do proteins like band 3, proteins 4.1R and 4.2, and adducin, which have potential binding partners in both complexes, sometimes switch allegiances? If so, is this a regulatory process? |
| How labile are the individual bonds in the membrane skeleton? Which bonds, other than the spectrin dimer-tetramer interaction, break when the skeleton is deformed? Are the rates of dissociation and reassociation such that the bonds dissociate during red cell deformation in the circulation? | |
| How is the skeleton disassembled and reassembled during invasion by malaria and other parasites? | |
| Lipids | What are the interactions between the membrane skeleton and the overlying lipids? Do these interactions have regulatory as well as structural functions? Does the membrane skeleton vary in unique regions of the lipid bilayer such as lipid rafts? |