A Highlights from MBoC Selection: Apical sorting of lysoGPI-anchored proteins occurs independent of association with detergent-resistant membranes but dependent on their N-glycosylation

Most glycosylphosphatidylinositol-anchored proteins (GPI-APs) are located at the apical surface of epithelial cells. The apical delivery of GPI-APs is believed to result from their association with lipid rafts. We find that overexpression of C-terminally tagged PGAP3 caused predominant production of lysoGPI-APs, an intermediate precursor in the GPI lipid remodeling process in Madin–Darby canine kidney cells. In these cells, produced lysoGPI-APs are not incorporated into detergent-resistant membranes (DRMs) but still are delivered apically, suggesting that GPI-AP association with DRMs is not necessary for apical targeting. In contrast, apical transport of both fully remodeled and lyso forms of GPI-APs is dependent on N-glycosylation, confirming a general role of N-glycans in apical protein transport. We also find that depletion of cholesterol causes apical-to-basolateral retargeting not only of fully remodeled GPI-APs, but also of lysoGPI-APs, as well as endogenous soluble and transmembrane proteins that would normally be targeted to the apical membrane. These findings confirm the essential role for cholesterol in the apical protein targeting and further demonstrate that the mechanism of cholesterol-dependent apical sorting is not related to DRM association of GPI-APs.     

Stoichiometry of complexes between mannose-binding protein and its associated serine proteases. Defining functional units for complement activation

Serum mannose-binding protein (MBP) initiates the lectin branch of the complement cascade by binding to sugars on the surfaces of microorganisms and activating two MBP-associated serine proteases (MASP-1 and MASP-2). Rat serum MBP consists of oligomers containing up to four copies of a subunit that is composed of three identical polypeptide chains. Biophysical analysis of intact and truncated MASPs indicates that each MASP is a homodimer that is stabilized through interactions involving an N-terminal CUB domain. The binding sites for MBP are formed from the three N-terminal MASP domains, in which two CUB modules interact with MBP. Each MASP dimer contains binding sites for two MBP subunits. Both sites must be occupied by subunits from a single MBP oligomer to form a stable complex. Thus, the smallest functional unit for complement activation consists of MBP dimers bound to MASP-1 or MASP-2 homodimers. Trimers and tetramers of MBP form complexes containing up to two MASPs. The results reveal how MASP-1 and MASP-2 can function independently to activate the complement cascade.     

Protein oligomerization modulates raft partitioning and apical sorting of GPI-anchored proteins

An essential but insufficient step for apical sorting of glycosylphosphatidylinositol (GPI)-anchored proteins (GPI-APs) in epithelial cells is their association with detergent-resistant microdomains (DRMs) or rafts. In this paper, we show that in MDCK cells both apical and basolateral GPI-APs associate with DRMs during their biosynthesis. However, only apical and not basolateral GPI-APs are able to oligomerize into high molecular weight complexes. Protein oligomerization begins in the medial Golgi, concomitantly with DRM association, and is dependent on protein-protein interactions. Impairment of oligomerization leads to protein missorting. We propose that oligomerization stabilizes GPI-APs into rafts and that this additional step is required for apical sorting of GPI-APs. Two alternative apical sorting models are presented.     

Characterization of a nonclathrin endocytic pathway: membrane cargo and lipid requirements

Clathrin-independent endocytosis internalizes plasma membrane proteins that lack cytoplasmic sequences recognized by clathrin adaptor proteins. There is evidence for different clathrin-independent pathways but whether they share common features has not been systematically tested. Here, we examined whether CD59, an endogenous glycosylphosphatidyl inositol-anchored protein (GPI-AP), and major histocompatibility protein class I (MHCI), an endogenous, integral membrane protein, entered cells through a common mechanism and followed a similar itinerary. At early times of internalization, CD59 and MHCI were found in the same Arf6-associated endosomes before joining clathrin cargo proteins such as transferrin in common sorting endosomes. CD59 and MHCI, but not transferrin, also were observed in the Arf6-associated tubular recycling membranes. Endocytosis of CD59 and MHCI required free membrane cholesterol because it was inhibited by filipin binding to the cell surface. Expression of active Arf6 stimulated endocytosis of GPI-APs and MHCI to the same extent and led to their accumulation in Arf6 endosomes that labeled intensely with filipin. This blocked delivery of GPI-APs and MHCI to early sorting endosomes and to lysosomes for degradation. Endocytosis of transferrin was not affected by any of these treatments. These observations suggest common mechanisms for endocytosis without clathrin.     

Implications of lipid moiety in oligomerization and immunoreactivities of GPI-anchored proteins

Glycosylphosphatidylinositol (GPI) enriches GPI-anchored proteins (GPI-AP) in lipid rafts by intimate interaction of its lipid moiety with sphingolipids and cholesterol. In addition to such lipid-lipid interactions, it has been reported that GPI may interact with protein moiety linked to GPI and affect protein conformations because GPI delipidation reduced immunoreactivities of protein. Here, we report that GPI-APs that have not undergone fatty acid remodeling exhibit reduced immunoreactivities in Western blotting, similar to delipidated proteins, compared with normal remodeled GPI-APs. In contrast, immunostaining in flow cytometry and immunoprecipitation did not show significant differences between remodeled and unremodeled GPI-APs. Moreover, detection with premixed primary/secondary antibody complexes or Fab fragments eliminated this difference in Western blotting. These results indicate that normally remodeled GPI enhanced oligomerization of GPI-APs and that inefficient oligomerization of unremodeled GPI-APs was responsible for reduced immunoreactivities. Moreover, the reduction in immunoreactivities of delipidated GPI-APs was most likely caused by the same effect. Finally, by chemical cross-linking of surface proteins in living cells and cell killing assay using a pore-forming bacterial toxin, we showed that enhanced oligomerization by GPI-remodeling occurs under a physiological membrane environment. Thus, this study clarifies the significance of GPI fatty acid remodeling in oligomerization of GPI-APs and provides useful information for technical studies of these cell components.     

Crystal structure of cell adhesion molecule nectin-2/CD112 and its binding to immune receptor DNAM-1/CD226

The nectin and nectin-like molecule (Necl) family includes important cell adhesion molecules (CAMs) characterized by their Ig-like nature. Such CAMs regulate a broad spectrum of cell-cell interactions, including the interaction between NK cells and cytotoxic T lymphocytes (CTLs) and their target cells. CAM members nectin-2 (CD112) and Necl-5 (CD155) are believed to form homodimers (for nectin-2) or heterodimers in their functions for cell adhesion, as well as to interact with immune costimulatory receptor DNAX accessory molecule 1 (DNAM-1) (CD226) to regulate functions of both NK and CTL cells. However, the structural basis of the interactive mode of DNAM-1 with nectin-2 or Necl-5 is not yet understood. In this study, a soluble nectin-2 Ig-like V-set domain (nectin-2v) was successfully prepared and demonstrated to bind to both soluble ectodomain and cell surface-expressed full-length DNAM-1. The 1.85-? crystal structure of nectin-2v displays a perpendicular homodimer arrangement, revealing the homodimer characteristics of the nectin and Necls. Further mutational analysis indicated that disruption of the homodimeric interface of nectin-2v led to a failure of the homodimer formation, as confirmed by crystal structure and biochemical properties of the mutant protein of nectin-2v. Interestingly, the monomer mutant also loses DNAM-1 binding, as evidenced by cell staining with tetramers and surface plasmon resonance assays. The data indicate that interaction with DNAM-1 requires either the homodimerization or engagement of the homodimeric interface of nectin-2v. These results have implications for immune intervention of tumors or autoimmune diseases in the DNAM-1/nectin-2-dependent pathway.     

Evidence for budding of human immunodeficiency virus type 1 selectively from glycolipid-enriched membrane lipid rafts

A number of recent studies have demonstrated the significance of detergent-insoluble, glycolipid-enriched membrane domains or lipid rafts, especially in regard to activation and signaling in T lymphocytes. These domains can be viewed as floating rafts composed of sphingolipids and cholesterol which sequester glycosylphosphatidylinositol (GPI)-linked proteins, such as Thy-1 and CD59. CD45, a 200-kDa transmembrane phosphatase protein, is excluded from these domains. We have found that human immunodeficiency virus type 1 (HIV-1) particles produced by infected T-cell lines acquire the GPI-linked proteins Thy-1 and CD59, as well as the ganglioside GM1, which is known to partition preferentially into lipid rafts. In contrast, despite its high expression on the cell surface, CD45 was poorly incorporated into virus particles. Confocal fluorescence microscopy revealed that HIV-1 proteins colocalized with Thy-1, CD59, GM1, and a lipid raft-specific fluorescent lipid, DiIC(16)(3), in uropods of infected Jurkat cells. CD45 did not colocalize with HIV-1 proteins and was excluded from uropods. Dot immunoassay of Triton X-100-extracted membrane fractions revealed that HIV-1 p17 matrix protein and gp41 were present in the detergent-resistant fractions and that [(3)H]myristic acid-labeled HIV Gag showed a nine-to-one enrichment in lipid rafts. We propose a model for the budding of HIV virions through lipid rafts whereby host cell cholesterol, sphingolipids, and GPI-linked proteins within these domains are incorporated into the viral envelope, perhaps as a result of preferential sorting of HIV Gag to lipid rafts.     

Binding and internalization of Helicobacter pylori VacA via cellular lipid rafts in epithelial cells

In this study we investigated the roles of lipid rafts and glycosylphosphatidylinositol-anchored proteins (GPI-APs) in the process of VacA binding and internalization into epithelial cells. Vacuolating activity analysis in AGS, CHO cells, and a CHO-derived line that highly expresses GPI-linked fasI proteins indicated the significance of cholesterol and GPI-APs for VacA activity. Flow cytometric analysis along with VacA-cholesterol co-extraction experiments showed a cholesterol-dependent manner for VacA cell-binding activity, while GPI-APs were not related to it. Differential detergent extraction and fractionation in sucrose density gradient showed co-association of VacA and fasI with rafts on cell membranes. Subcellular distribution of fasI visualized by confocal microscope suggested that fasI trafficked via a newly defined endocytic pathway for GPI-APs in the derived line. Upon VacA intoxication, VacA was visualized to co-migrate along with fasI and finally induced vacuolation coupled with dramatic redistribution of fasI molecules. These results suggest that VacA exploits rafts for docking and entering the cell via the endocytic pathway of GPI-APs.     

Structural characterization and unfolding mechanism of human 4F2hc ectodomain

4F2hc (CD98hc) is a multifunctional type II membrane glycoprotein involved in several functions as amino acid transport, cell fusion, β1-integrin-signaling and transformation. 4F2hc ectodomain has been crystallized and its three-dimensional structure determined. We have carried out a spectroscopical/structural characterization of the recombinant ectodomain in order to obtain information on its dynamic structure in solution and on its ability to form homodimers by itself in the absence of the transmembrane helix and of the potential interactions with the plasma membrane. Analytical ultracentrifugation and crosslinking experiments showed that the ectodomain is monomeric in solution. The secondary structure determined by far-UV circular dichroism (CD) spectroscopy (around 30% α-helix and 20% β-sheets, 12% antiparallel and 8% parallel) reveals a compact and thermally stable structure with a high melting temperature (57-59°C). Tryptophan residues are mainly buried and immobilized in the hydrophobic core of the protein as suggested by near-UV CD spectrum, the position of the Trp maximum fluorescence emission (323nm) and from the acrylamide quenching constant (2.6M(-1)). Urea unfolding equilibrium has been studied by far-UV CD and fluorescence spectroscopy to gain information on the folding/unfolding process of the ectodomain. The analyses suggest the existence of two intermediate states as reported for other TIM barrel-containing proteins rather than an independent unfolding of each domain [A, (βα)(8) barrel; C, antiparallel β(8) sandwich]. Folding seems to be directed by the initial formation of hydrophobic clusters within the first strands of the β-barrel of domain A followed by additional hydrophobic interactions in domain C.     

Enhanced response of T lymphocytes from Pgap3 knockout mouse: Insight into roles of fatty acid remodeling of GPI anchored proteins

Glycosylphosphatidylinositol (GPI) is a complex glycolipid that serves as a membrane anchor for many cell-surface proteins, such as Thy-1 and CD48. GPI-anchored proteins (GPI-APs) play important roles in many biological processes, such as signal transduction and cell-cell interaction, through their association with lipid rafts. Fatty acid remodeling of GPI-APs in the Golgi apparatus is required for their efficient association with lipid rafts, i.e., the unsaturated fatty acid at the sn-2 position of the PI moiety is exchanged for the saturated fatty acid by PGAP2 and PGAP3. To investigate the immunological role of the fatty acid remodeling of GPI-APs, we generated a Pgap3 knockout mouse. In this mouse, GPI-APs are expressed on the cell surface without fatty acid remodeling, and fail to associate with lipid rafts. Male Pgap3 knockout mice were born alive at a ratio lower than expected from Mendel's law, whereas the number of female mice followed Mendel's law. All mice exhibited growth retardation and abnormal reflexes such as limb grasping. We focused T cell function in these mice and found that T cell development in the absence of Pgap3 was normal. However, the response of T cells was enhanced in Pgap3 knockout mice in both in vitro and in vivo studies, including alloreactive response, antigen-specific immune response, and experimental autoimmune encephalomyelitis. Cross-linking of Thy-1 in wild-type cells inhibited the signal transduced by the T cell receptor (TCR), whereas cross-linking of Thy-1 in Pgap3 knockout cells enhanced the TCR signal. These results suggest that GPI-APs localized in lipid rafts may modulate signaling through the TCR.     

Endocytosis of glycosylphosphatidylinositol-anchored proteins

Glycosylphosphatidylinositol-anchored proteins (GPI-APs) represent an interesting amalgamation of the three basic kinds of cellular macromolecules viz. proteins, carbohydrates and lipids. An unusually hybrid moiety, the GPI-anchor is expressed in a diverse range of organisms from parasites to mammalian cells and serves to anchor a large number of functionally diverse proteins and has been the center of attention in scientific debate for some time now. Membrane organization of GPI-APs into laterally-organized cholesterol-sphingolipid ordered membrane domains or "rafts" and endocytosis of GPI-APs has been intensely debated. Inclusion into or exclusion from these membrane domains seems to be the critical factor in determining the endocytic mechanisms and intracellular destinations of GPI-APs. The intracellular signaling as well as endocytic trafficking of GPI-APs is critically dependent upon the cell surface organization of GPI-APs, and the associations with these lipid rafts play a vital role during these processes. The mechanism of endocytosis for GPI-APs may differ from other cellular endocytic pathways, such as those mediated by clathrin-coated pits (caveolae), and is necessary for unique biological functions. Numerous intracellular factors are involved in and regulate the endocytosis of GPI-APs, and these may be variably dependent on cell-type. The central focus of this article is to describe the significance of the endocytosis of GPI-APs on a multitude of biological processes, ranging from nutrient-uptake to more complex immune responses. Ultimately, a thorough elucidation of GPI-AP mediated signaling pathways and their regulatory elements will enhance our understanding of essential biological processes and benefit as components of disease intervention strategies.     

The fusion peptide of Semliki Forest virus associates with sterol-rich membrane domains

Semliki Forest virus (SFV) is an enveloped alphavirus whose membrane fusion is triggered by low pH and promoted by cholesterol and sphingolipid in the target membrane. Fusion is mediated by E1, a viral membrane protein containing the putative fusion peptide. Virus mutant studies indicate that SFV's cholesterol dependence is controlled by regions of E1 outside of the fusion peptide. Both E1 and E1*, a soluble ectodomain form of E1, interact with membranes in a reaction dependent on low pH, cholesterol, and sphingolipid and form highly stable homotrimers. Here we have used detergent extraction and gradient floatation experiments to demonstrate that E1* associated selectively with detergent-resistant membrane domains (DRMs or rafts). In contrast, reconstituted full-length E1 protein or influenza virus fusion peptide was not associated with DRMs. Methyl beta-cyclodextrin quantitatively extracted both cholesterol and E1* from membranes in the absence of detergent, suggesting a strong association of E1* with sterol. Monoclonal antibody studies demonstrated that raft association was mediated by the proposed E1 fusion peptide. Thus, although other regions of E1 are implicated in the control of virus cholesterol dependence, once the SFV fusion peptide inserts in the target membrane it has a high affinity for membrane domains enriched in cholesterol and sphingolipid.     

Cyclodextrins but not compactin inhibit the lateral diffusion of membrane proteins independent of cholesterol

Cholesterol and glycosphingolipid-enriched membrane domains, termed lipid rafts, were proposed to play important roles in trafficking and signaling events. These functions are inhibited following putative disruption of rafts by cholesterol depletion, commonly induced by treatment with methyl-beta-cyclodextrin (MbetaCD). However, several studies showed that the lateral diffusion of membrane proteins is inhibited by MbetaCD, suggesting that it may have additional effects on membrane organization unrelated to cholesterol removal. Here, we investigated this possibility by comparison of the effects of cholesterol depletion by MbetaCD and by metabolic inhibition (compactin), and of treatment with alpha-CD, which does not bind cholesterol. The studies employed two series of proteins (Ras and influenza hemagglutinin), each containing as internal controls related mutants that differ in raft association. Mild MbetaCD treatment retarded the lateral diffusion of both raft and non-raft mutants, whereas similar cholesterol reduction (30-33%) by metabolic inhibition enhanced selectively the diffusion of the raft-associated mutants. Moreover, alpha-CD also inhibited the diffusion of raft and non-raft mutants, despite its lack of effect on cholesterol content. These findings suggest that the widely used treatment with CD to reduce cholesterol has additional, cholesterol-independent effects on membrane protein mobility, which do not necessarily distinguish between raft and non-raft proteins.     

Nanoscale organization of multiple GPI-anchored proteins in living cell membranes

Cholesterol and sphingolipid-enriched "rafts" have long been proposed as platforms for the sorting of specific membrane components including glycosyl-phosphatidylinositol-anchored proteins (GPI-APs), however, their existence and physical properties have been controversial. Here, we investigate the size of lipid-dependent organization of GPI-APs in live cells, using homo and hetero-FRET-based experiments, combined with theoretical modeling. These studies reveal an unexpected organization wherein cell surface GPI-APs are present as monomers and a smaller fraction (20%-40%) as nanoscale (<5 nm) cholesterol-sensitive clusters. These clusters are composed of at most four molecules and accommodate diverse GPI-AP species; crosslinking GPI-APs segregates them from preexisting GPI-AP clusters and prevents endocytosis of the crosslinked species via a GPI-AP-selective pinocytic pathway. In conjunction with an analysis of the statistical distribution of the clusters, these observations suggest a mechanism for functional lipid-dependent clustering of GPI-APs.     

Examination of Sec22 Homodimer Formation and Role in SNARE-dependent Membrane Fusion

Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein complexes play essential roles in catalyzing intracellular membrane fusion events although the assembly pathway and molecular arrangement of SNARE complexes in membrane fusion reactions are not well understood. Here we monitored interactions of the R-SNARE protein Sec22 through a cysteine scanning approach and detected efficient formation of cross-linked Sec22 homodimers in cellular membranes when cysteine residues were positioned in the SNARE motif or C terminus of the transmembrane domain. When specific Sec22 cysteine derivatives are present on both donor COPII vesicles and acceptor Golgi membranes, the formation of disulfide cross-links provide clear readouts on trans- and cis-SNARE arrangements during this fusion event. The Sec22 transmembrane domain was required for efficient homodimer formation and for membrane fusion suggesting a functional role for Sec22 homodimers. We propose that Sec22 homodimers promote assembly of higher-order SNARE complexes to catalyze membrane fusion. Sec22 is also reported to function in macroautophagy and in formation of endoplasmic reticulum-plasma membrane contact sites therefore homodimer assembly may regulate Sec22 activity across a range of cellular processes.     

Depletion of cellular cholesterol and lipid rafts increases shedding of CD30

CD30, a lymphoid activation marker, is shed into the cell environment after endoproteolytic cleavage of its ectodomain. Soluble (s)CD30 is able to suppress the Th1-type immune response. Because high serum levels of sCD30 and cholesterol-lowering drugs seem to be beneficial in some Th1-type autoimmune diseases, we focused on a link between CD30 shedding and the amount of cellular cholesterol. Cholesterol depletion of human Hodgkin lymphoma- and non-Hodgkin lymphoma-derived cell lines by methyl-beta-cyclodextrin led to a down-regulation of membrane-bound CD30 and increased release of sCD30. Additionally, the cholesterol-interfering drugs lovastatin, cholesterol oxidase, and filipin increased CD30 shedding. Both the down-regulation of membrane-anchored CD30 and the release of sCD30 were dependent on metalloproteinases. Using specific inhibitors, we detected TNF-alpha converting enzyme (TACE) as the leading enzyme responsible for cholesterol-dependent CD30 shedding. A Triton X-100-based method for lipid raft isolation revealed that CD30 was partially present in lipid rafts, whereas TACE was localized in the nonraft fractions. Disintegration of lipid rafts by cholesterol depletion might therefore lead to dynamic interactions of CD30 with TACE, resulting in enhanced shedding of CD30. Our results suggest a possible role of cholesterol-dependent shedding of CD30 in the pathogenesis of immune diseases.     

Glycosylphosphatidylinositol-anchored proteins as regulators of cortical cytoskeleton

Glycosylphosphatidylinositol-anchored proteins (GPI-AP) are important players in reception and signal transduction, cell adhesion, guidance, formation of immune synapses, and endocytosis. At that, a particular GPI-AP can have different activities depending on a ligand. It is known that GPI-AP oligomer creates a lipid raft in its base on plasma membrane, which serves as a signaling platform for binding and activation of src-family kinases. Yet, this does not explain different activities of GPI-APs. Meanwhile, it has been shown that short-lived actomyosin complexes are bound to GPI-APs through lipid rafts. Here, we hypothesize that cell cortical cytoskeleton is the main target of GPI-AP signaling. Our hypothesis is based on the fact that the GPI-AP-induced lipid raft bound to actin filaments and anionic lipids of this raft is known to interact with and activate various actin-nucleating factors, such as formins and N-WASP. It is also known that these and other actin-regulating proteins are activated by src-family kinases directly or through their effectors, such as cortactin and abl-kinases. Regulation of cytoskeleton by GPI-APs may have impact on morphogenesis, cell guidance, and endocytosis, as well as on signaling of other receptors. To evaluate our hypothesis, we have comprehensively considered physiological activities of two GPI-APs–urokinase receptor and T-cadherin.     

Isolation and characterization of sea urchin egg lipid rafts and their possible function during fertilization.

Specialized membrane microdomains called rafts are thought to play a role in many types of cell-cell interactions and signaling. We have investigated the possibility that sea urchin eggs contain these specialized membrane microdomains and if they play a role in signal transduction at fertilization. A low density, TX-100 insoluble membrane fraction, typical of lipid rafts, was isolated by equilibrium gradient centrifugation. This raft fraction contained proteins distinct from cytoskeletal complexes. The fraction was enriched in tyrosine phosphorylated proteins and contained two proteins known to be involved in signaling during egg activation (an egg Src-type kinase and PLC gamma). This fraction was further characterized as a prototypical raft fraction by the release of proteins in response to in vitro treatment of the rafts with the cholesterol binding drug, methyl-beta-cyclodextrin (M beta CD). Furthermore, treatment of eggs with M beta CD inhibited fertilization, suggesting that egg lipid rafts play a physiological role in fertilization. Mol. Reprod. Dev. 59:294-305, 2001.     

Transient homodimer interactions studied using the electron self-exchange reaction

Transient homodimer protein interactions have been investigated by analyzing the influence of ionic strength (NaCl) on the electron self-exchange (the bimolecular reaction whereby the two oxidation states of a redox protein interconvert) rate constant (k(ese)) of four plastocyanins. The k(ese) values for the plastocyanins from spinach, Dryopteris crassirhizoma (a fern), and the green alga Ulva pertusa, which possess acidic patches of varying size and locations, increase 190-, 29-, and 21-fold, respectively, at elevated ionic strength (I = 2.03 M). In contrast, the k(ese) for the almost neutral cyanobacterial plastocyanin from Anabaena variabilis exhibits very little dependence on ionic strength. The temperature dependence of the k(ese) for spinach plastocyanin (I = 0.28 M) provides evidence for poor packing at the homodimer interface. Representative structures of the transient homodimers involved in electron self-exchange, which are consistent with fits of the ionic strength dependence of k(ese) to van Leeuwen theory, have been obtained from protein modeling and docking simulations. The Coulombic energy of the docked homodimers follows the order spinach > D. crassirhizoma > U. pertusa > A. variabilis, which matches that of the overall influence of ionic strength on k(ese). Analysis of the homodimer structures indicates that poor packing and high planarity are features of the interface that favor transient interactions. The physiologically relevant Mg2+ ion has a much more pronounced influence on the k(ese) of spinach plastocyanin, which along with the known properties of the thylakoid lumen suggests a biological role for electron self-exchange.