Occludin OCEL-domain interactions are required for maintenance and regulation of the tight junction barrier to macromolecular flux

In vitro and in vivo studies implicate occludin in the regulation of paracellular macromolecular flux at steady state and in response to tumor necrosis factor (TNF). To define the roles of occludin in these processes, we established intestinal epithelia with stable occludin knockdown. Knockdown monolayers had markedly enhanced tight junction permeability to large molecules that could be modeled by size-selective channels with radii of ∼62.5 Å. TNF increased paracellular flux of large molecules in occludin-sufficient, but not occludin-deficient, monolayers. Complementation using full-length or C-terminal coiled-coil occludin/ELL domain (OCEL)–deficient enhanced green fluorescent protein (EGFP)–occludin showed that TNF-induced occludin endocytosis and barrier regulation both required the OCEL domain. Either TNF treatment or OCEL deletion accelerated EGFP-occludin fluorescence recovery after photobleaching, but TNF treatment did not affect behavior of EGFP-occludin^ΔOCEL. Further, the free OCEL domain prevented TNF-induced acceleration of occludin fluorescence recovery, occludin endocytosis, and barrier loss. OCEL mutated within a recently proposed ZO-1–binding domain (K433) could not inhibit TNF effects, but OCEL mutated within the ZO-1 SH3-GuK–binding region (K485/K488) remained functional. We conclude that OCEL-mediated occludin interactions are essential for limiting paracellular macromolecular flux. Moreover, our data implicate interactions mediated by the OCEL K433 region as an effector of TNF-induced barrier regulation.Tight junctions seal the paracellular space in simple epithelia, such as those lining the lungs, intestines, and kidneys (Anderson et al., 2004 ; Fanning and Anderson, 2009 ; Shen et al., 2011 ). In the intestine, reduced paracellular barrier function is associated with disorders in which increased paracellular flux of ions and molecules contributes to symptoms such as diarrhea, malabsorption, and intestinal protein loss. Recombinant tumor necrosis factor (TNF) can be used to model this barrier loss in vitro or in vivo (Taylor et al., 1998 ; Clayburgh et al., 2006 ), and TNF neutralization is associated with restoration of intestinal barrier function in Crohn''s disease (Suenaert et al., 2002 ). Further, in vivo and in vitro studies of intestinal epithelia show that TNF-induced barrier loss requires myosin light chain kinase (MLCK) activation (Zolotarevsky et al., 2002 ; Clayburgh et al., 2005 , 2006 ; Ma et al., 2005 ; Wang et al., 2005 ). The resulting myosin II regulatory light chain (MLC) phosphorylation drives occludin internalization, which is required for cytokine-induced intestinal epithelial barrier loss (Clayburgh et al., 2005 , 2006 ; Schwarz et al., 2007 ; Marchiando et al., 2010 ). In addition, transgenic EGFP-occludin expression in vivo limits TNF-induced depletion of tight junction–associated occludin, barrier loss, and diarrhea (Marchiando et al., 2010 ). Conversely, in vitro studies show that occludin knockdown limits TNF-induced barrier regulation (Van Itallie et al., 2010 ). The basis for this discrepancy is not understood.One challenge is that, despite being identified 20 yr ago (Furuse et al., 1993 ), the contribution of occludin to tight junction regulation remains incompletely defined. The observation that occludin-knockout mice are able to form paracellular barriers and do not have obvious defects in epidermal, respiratory, or bladder tight junction function (Saitou et al., 2000 ; Schulzke et al., 2005 ) led many to conclude that occludin is not essential for tight junction barrier function. It is important to note, however, that barrier regulation in response to stress has not been studied in occludin-deficient animals.We recently showed that dephosphorylation of occludin serine-408 promotes assembly of a complex composed of occludin, ZO-1, and claudin-2 that inhibits flux across size- and charge-selective channels termed the pore pathway (Anderson and Van Itallie, 2009 ; Turner, 2009 ; Raleigh et al., 2011 ; Shen et al., 2011 ). Although this demonstrates that occludin can serve a regulatory role, it does not explain the role of occludin in TNF-induced barrier loss, which increases flux across the size- and charge-nonselective leak pathway (Wang et al., 2005 ; Weber et al., 2010 ). In vitro studies, however, do suggest that occludin contributes to leak pathway regulation, as occludin knockdown in either Madin–Darby canine kidney (MDCK) or human intestinal (Caco-2) epithelial monolayers enhances leak pathway permeability (Yu et al., 2005 ; Al-Sadi et al., 2011 ; Ye et al., 2011 ). Taken as a whole, these data suggest that occludin organizes the tight junction to limit leak pathway flux, whereas occludin removal, either by knockdown or endocytosis, enhances leak pathway flux.To define the mechanisms by which occludin regulates the leak pathway, we analyzed the contributions of occludin, as well as specific occludin domains, to basal and TNF-induced barrier regulation. The data indicate that TNF destabilizes tight junction–associated occludin via interactions mediated by the C-terminal coiled-coil occludin/ELL domain (OCEL). Further, these OCEL-mediated events are required for TNF-induced barrier regulation. Thus these data provide new insight into the structural elements and mechanisms by which occludin regulates leak pathway paracellular flux.     

Insulin responsiveness of glucose transporter 4 in 3T3-L1 cells depends on the presence of sortilin

Insulin-dependent translocation of glucose transporter 4 (Glut4) to the plasma membrane of fat and skeletal muscle cells plays the key role in postprandial clearance of blood glucose. Glut4 represents the major cell-specific component of the insulin-responsive vesicles (IRVs). It is not clear, however, whether the presence of Glut4 in the IRVs is essential for their ability to respond to insulin stimulation. We prepared two lines of 3T3-L1 cells with low and high expression of myc₇-Glut4 and studied its translocation to the plasma membrane upon insulin stimulation, using fluorescence-assisted cell sorting and cell surface biotinylation. In undifferentiated 3T3-L1 preadipocytes, translocation of myc₇-Glut4 was low regardless of its expression levels. Coexpression of sortilin increased targeting of myc₇-Glut4 to the IRVs, and its insulin responsiveness rose to the maximal levels observed in fully differentiated adipocytes. Sortilin ectopically expressed in undifferentiated cells was translocated to the plasma membrane regardless of the presence or absence of myc₇-Glut4. AS160/TBC1D4 is expressed at low levels in preadipocytes but is induced in differentiation and provides an additional mechanism for the intracellular retention and insulin-stimulated release of Glut4.Adipocytes, skeletal muscle cells, and some neurons respond to insulin stimulation by translocating intracellular glucose transporter 4 (Glut4) to the plasma membrane. In all these cells, the insulin-responsive pool of Glut4 is localized in small membrane vesicles, the insulin-responsive vesicles (IRVs; Kandror and Pilch, 2011 ; Bogan, 2012 ). The protein composition of these vesicles has been largely characterized (Kandror and Pilch, 2011 ; Bogan, 2012 ). The IRVs consist predominantly of Glut4, insulin-responsive aminopeptidase (IRAP), sortilin, low-density-lipoprotein receptor–related protein 1 (LRP1), SCAMPs, and VAMP2. Glut4, IRAP, and sortilin physically interact with each other, which might be important for the biogenesis of the IRVs (Shi and Kandror, 2007 ; Shi et al., 2008 ). In addition, the IRVs compartmentalize recycling receptors, such as the transferrin receptor and the IGF2/mannose 6-phosphate receptor, although it is not clear whether these receptors represent obligatory vesicular components or their presence in the IRVs is explained by mass action (Pilch, 2008 ), inefficient sorting, or other reasons.Deciphering of the protein composition of the IRVs is important because it is likely to explain their unique functional property: translocation to the plasma membrane in response to insulin stimulation. Even if we presume that IRV trafficking is controlled by loosely associated peripheral membrane proteins, the latter should still somehow recognize the core vesicular components that create the “biochemical individuality” of this compartment. In spite of our knowledge of the IRV protein composition, however, the identity of the protein(s) that confer insulin sensitivity to these vesicles is unknown.Insulin responsiveness of the IRVs was associated with either IRAP or Glut4. Thus it was shown that Glut4 interacted with the intracellular anchor TUG (Bogan et al., 2003 , 2012 ), whereas IRAP associated with other proteins implemented in the regulation of Glut4 translocation, such as AS160 (Larance et al., 2005 ; Peck et al., 2006 ), p115 (Hosaka et al., 2005 ), tankyrase (Yeh et al., 2007 ), and several others (reviewed in Bogan, 2012 ). Results of these studies, or at least their interpretations, are not necessarily consistent with each other, as the existence of multiple independent anchors for the IRVs is, although possible, unlikely.Ablation of the individual IRV proteins has also led to controversial data. Thus knockout of IRAP decreases total protein levels of Glut4 but does not affect its translocation in the mouse model (Keller et al., 2002 ). On the contrary, knockdown of IRAP in 3T3-L1 adipocytes has a strong inhibitory effect on translocation of Glut4 (Yeh et al., 2007 ). In yet another study, knockdown of IRAP in 3T3-L1 adipocytes did not affect insulin-stimulated translocation of Glut4 but increased its plasma membrane content under basal conditions (Jordens et al., 2010 ). By the same token, total or partial ablation of Glut4 had various effects on expression levels, intracellular localization, and translocation of IRAP (Jiang et al., 2001 ; Abel et al., 2004 ; Carvalho et al., 2004 ; Gross et al., 2004 ; Yeh et al., 2007 ). Knockdown of either sortilin or LRP1 decreased protein levels of Glut4 (Shi and Kandror, 2005 ; Jedrychowski et al., 2010 ).One model that might explain these complicated and somewhat inconsistent results is that depletion of either major integral protein of the IRVs disrupts the network of interactions between vesicular proteins and thus decreases the efficiency of protein sorting into the IRVs (Kandror and Pilch, 2011 ). Correspondingly, the remaining IRV components that cannot be faithfully compartmentalized in the vesicles are either degraded (Jiang et al., 2001 ; Keller et al., 2002 ; Abel et al., 2004 ; Carvalho et al., 2004 ; Shi and Kandror, 2005 ; Yeh et al., 2007 ; Jedrychowski et al., 2010 ) or mistargeted (Jiang et al., 2001 ; Jordens et al., 2010 ), depending on experimental conditions and types of cells used in these studies. In other words, knockdown of any major IRV component may decrease vesicle formation along with insulin responsiveness. Thus, in spite of a large body of literature, the identity of protein(s) that confer insulin responsiveness to the IRVs is unknown.Here we used a gain-of-function approach to address this question. Specifically, we attempted to “build” functional IRVs in undifferentiated 3T3-L1 preadipocytes by forced expression of the relevant proteins. Undifferentiated preadipocytes do not express Glut4 or sortilin and lack IRVs (ElJack et al., 1999 ; Shi and Kandror, 2005 ; Shi et al., 2008 ). Correspondingly, IRAP, which is expressed in these cells, shows low insulin response (Ross et al., 1998 ; Shi et al., 2008 ). We found that ectopic expression of increasing amounts of Glut4 in undifferentiated preadipocytes does not lead to its marked translocation to the plasma membrane upon insulin stimulation. On the contrary, sortilin expressed in undifferentiated preadipocytes was localized in the IRVs and was translocated to the plasma membrane in response to insulin stimulation. Moreover, upon coexpression with Glut4, sortilin dramatically increased its insulin responsiveness to the levels observed in fully differentiated adipocytes. Thus sortilin may represent the key component of the IRVs, which is responsible not only for the formation of vesicles (Shi and Kandror, 2005 ; Ariga et al., 2008 ; Hatakeyama and Kanzaki, 2011 ), but also for their insulin responsiveness. It is worth noting that sortilin levels are significantly decreased in obese and diabetic humans and mice (Kaddai et al., 2009 ). We thus suggest that sortilin may be a novel and important target in the fight against insulin resistance and diabetes.Our experiments also demonstrate that undifferentiated preadipocytes lack a mechanism for the full intracellular retention of Glut4 that can be achieved by ectopic expression of AS160/TBC1D4.     

Phosphorylation Dependence of Hsp27 Multimeric Size and Molecular Chaperone Function

The molecular chaperone Hsp27 exists as a distribution of large oligomers that are disassembled by phosphorylation at Ser-15, -78, and -82. It is controversial whether the unphosphorylated Hsp27 or the widely used triple Ser-to-Asp phospho-mimic mutant is the more active molecular chaperone in vitro. This question was investigated here by correlating chaperone activity, as measured by the aggregation of reduced insulin or α-lactalbumin, with Hsp27 self-association as monitored by analytical ultracentrifugation. Furthermore, because the phospho-mimic is generally assumed to reproduce the phosphorylated molecule, the size and chaperone activity of phosphorylated Hsp27 were compared with that of the phospho-mimic. Hsp27 was triply phosphorylated by MAPKAP-2 kinase, and phosphorylation was tracked by urea-PAGE. An increasing degree of suppression of insulin or α-lactalbumin aggregation correlated with a decreasing Hsp27 self-association, which was the least for phosphorylated Hsp27 followed by the mimic followed by the unphosphorylated protein. It was also found that Hsp27 added to pre-aggregated insulin did not reverse aggregation but did inhibit these aggregates from assembling into even larger aggregates. This chaperone activity appears to be independent of Hsp27 phosphorylation. In conclusion, the most active chaperone of insulin and α-lactalbumin was the Hsp27 (elongated) dimer, the smallest Hsp27 subunit observed under physiological conditions. Next, the Hsp27 phospho-mimic is only a partial mimic of phosphorylated Hsp27, both in self-association and in chaperone function. Finally, the efficient inhibition of insulin aggregation by Hsp27 dimer led to the proposal of two models for this chaperone activity.Oligomeric heat shock protein 27 (Hsp27)² is a ubiquitous mammalian protein with a variety of functions in health and disease (1–8). These functions include ATP-independent chaperone activity in response to environmental stress, e.g. heat shock and oxidative stress, control of apoptosis, and regulation of actin cytoskeleton dynamics. Hsp27 is a member of the α-crystallin small heat shock protein family of which αB-crystallin is the archetype. These proteins are characterized by an α-crystallin domain of 80–90 residues consisting of roughly eight β-strands that form an intermolecular β-sheet interaction interface within a dimer, the basic building subunit of the oligomer (2, 4, 9–11).Hsp27 is in equilibrium between high molecular weight oligomers and much lower molecular weight multimers. It has been reported that unphosphorylated Hsp27 includes predominantly a distribution of high molecular species ranging in size from 12-mer to 35-mer (12–19). Phosphorylation of Hsp27 at serines 15, 78, and 82 by the p38-activated MAPKAP-2 kinase (20–22) or the use of the triple Ser-to-Asp phospho-mimic results in a major shift in the equilibrium toward much smaller multimers (23) and in an alteration of its function (1, 3, 6, 7, 24, 25). The size distribution of the smaller species has been reported to be between monomer and tetramer (12–16, 18, 19).Small heat shock proteins, including Hsp27, behave as ATP-independent molecular chaperones during cellular heat shock. They bind partially unfolded proteins and prevent their aggregation until the proteins can be refolded by larger ATP-dependent chaperones or are digested (7, 8, 26). This function includes the up-regulation and/or phosphorylation of Hsp27.It is not entirely clear what the role of Hsp27 size and phosphorylation state plays in its heat shock function because there are conflicting results in the literature. Some in vitro studies concluded that the unphosphorylated oligomeric Hsp27 (or the murine isoform Hsp25) protects proteins against aggregation better than does the phosphorylation mimic (13, 19, 27), whereas others found no difference (16, 28, 29), and still other studies found that the mimic protects better than does the unphosphorylated wild type (27, 30, 31). In-cell studies found that phosphorylation of Hsp27 was essential for thermo-protection of actin filaments (32), and the Hsp27 phosphorylation mimic decreased inclusion body formation better than did unphosphorylated Hsp27 (33). This study was undertaken to investigate the molecular chaperone function of Hsp27 by correlating chaperone activity with Hsp27 size and by comparing fully phosphorylated Hsp27 with its phospho-mimic.     

ERS-24, a Mammalian v-SNARE Implicated in Vesicle Traffic between the ER and the Golgi

We report the identification and characterization of ERS-24 (Endoplasmic Reticulum SNARE of 24 kD), a new mammalian v-SNARE implicated in vesicular transport between the ER and the Golgi. ERS24 is incorporated into 20S docking and fusion particles and disassembles from this complex in an ATP-dependent manner. ERS-24 has significant sequence homology to Sec22p, a v-SNARE in Saccharomyces cerevisiae required for transport between the ER and the Golgi. ERS-24 is localized to the ER and to the Golgi, and it is enriched in transport vesicles associated with these organelles.Newly formed transport vesicles have to be selectively targeted to their correct destinations, implying the existence of a set of compartment-specific proteins acting as unique receptor–ligand pairs. Such proteins have now been identified (Söllner et al., 1993a ; Rothman, 1994): one partner efficiently packaged into vesicles, termed a v-SNARE,¹ and the other mainly localized to the target compartment, a t-SNARE. Cognate pairs of v- and t-SNAREs, capable of binding each other specifically, have been identified for the ER–Golgi transport step (Lian and Ferro-Novick, 1993; Søgaard et al., 1994), the Golgi–plasma membrane transport step (Aalto et al., 1993; Protopopov et al., 1993; Brennwald et al., 1994) in Saccharomyces cerevisiae, and regulated exocytosis in neuronal synapses (Söllner et al., 1993a ; for reviews see Scheller, 1995; Südhof, 1995). Additional components, like p115, rab proteins, and sec1 proteins, appear to regulate vesicle docking by controlling the assembly of SNARE complexes (Søgaard et al., 1994; Lian et al., 1994; Sapperstein et al., 1996; Hata et al., 1993; Pevsner et al., 1994).In contrast with vesicle docking, which requires compartment-specific components, the fusion of the two lipid bilayers uses a more general machinery derived, at least in part, from the cytosol (Rothman, 1994), which includes an ATPase, the N-ethylmaleimide–sensitive fusion protein (NSF) (Block et al., 1988; Malhotra et al., 1988), and soluble NSF attachment proteins (SNAPs) (Clary et al., 1990; Clary and Rothman, 1990; Whiteheart et al., 1993). Only the assembled v–t-SNARE complex provides high affinity sites for the consecutive binding of three SNAPs (Söllner et al., 1993b ; Hayashi et al., 1995) and NSF. When NSF is inactivated in vivo, v–t-SNARE complexes accumulate, confirming that NSF is needed for fusion after stable docking (Søgaard et al., 1994).The complex of SNAREs, SNAPs, and NSF can be isolated from detergent extracts of cellular membranes in the presence of ATPγS, or in the presence of ATP but in the absence of Mg²⁺, and sediments at ∼20 Svedberg (20S particle) (Wilson et al., 1992). In the presence of MgATP, the ATPase of NSF disassembles the v–t-SNARE complex and also releases SNAPs. It seems likely that this step somehow initiates fusion.To better understand vesicle flow patterns within cells, it is clearly of interest to identify new SNARE proteins. Presently, the most complete inventory is in yeast, but immunolocalization is difficult in yeast compared with animal cells, and many steps in protein transport have been reconstituted in animal extracts (Rothman, 1992) that have not yet been developed in yeast. Therefore, it is important to create an inventory of SNARE proteins in animal cells. The most unambiguous and direct method for isolating new SNAREs is to exploit their ability to assemble together with SNAPs and NSF into 20S particles and to disassemble into subunits when NSF hydrolyzes ATP. Similar approaches have already been successfully used to isolate new SNAREs implicated in ER to Golgi (Søgaard et al., 1994) and intra-Golgi transport (Nagahama et al., 1996), in addition to the original discovery of SNAREs in the context of neurotransmission (Söllner et al., 1993a ).Using this method, we now report the isolation and detailed characterization of ERS-24 (Endoplasmic Reticulum SNARE of 24 kD), a new mammalian v-SNARE that is localized to the ER and Golgi. ERS-24 is found in transport vesicles associated with the transitional areas of the ER and with the rims of Golgi cisternae, suggesting a role for ERS-24 in vesicular transport between these two compartments.     

Molecular Chaperone Hsp70/Hsp90 Prepares the Mitochondrial Outer Membrane Translocon Receptor Tom71 for Preprotein Loading

The preproteins targeted to the mitochondria are transported through the translocase of the outer membrane complex. Tom70/Tom71 is a major surface receptor of the translocase of the outer membrane complex for mitochondrial preproteins. The preproteins are escorted to Tom70/Tom71 by molecular chaperones Hsp70 and Hsp90. Here we present the high resolution crystal structures of Tom71 and the protein complexes between Tom71 and the Hsp70/Hsp90 C terminus. The crystal structures indicate that Tom70/Tom71 may exhibit two distinct states. In the closed state, the N-terminal domain of Tom70/Tom71 partially blocks the preprotein-binding pocket. In the open state, the N-terminal domain moves away, and the preprotein-binding pocket is fully exposed. The complex formation between the C-terminal EEVD motif of Hsp70/Hsp90 and Tom71 could lock Tom71 in the open state where the preprotein-binding pocket of Tom71 is ready to receive preproteins. The interactions between Hsp70/Hsp90 and Tom71 N-terminal domain generate conformational changes that may increase the volume of the preprotein-binding pocket. The complex formation of Hsp70/Hsp90 and Tom71 also generates significant domain rearrangement within Tom71, which may position the preprotein-binding pocket closer to Hsp70/Hsp90 to facilitate the preprotein transfer from the molecular chaperone to Tom71. Therefore, molecular chaperone Hsp70/Hsp90 may function to prepare the mitochondrial outer membrane receptor Tom71 for preprotein loading.The mitochondrion plays important roles in cell physiology. The mitochondrion functions as the “cellular power house” by generating most of the supply of ATP for the cell. In addition, the mitochondrion is involved in a number of critical cellular processes including the synthesis of metabolites, lipid metabolism, free radical production, and metal ion homeostasis. The mitochondrion consists of four compartments, the outer membrane, the inner membrane, the intermembrane space, and the mitochondrial matrix. The mitochondrion contains a large number of proteins (1), but only a few of these are translated within the mitochondrion (2). Therefore, the majority of the mitochondrial proteins are synthesized in the cytosol and translocated into the mitochondrion.The mitochondrial preproteins contain specific targeting signals to reach the correct compartments within the mitochondria. The mitochondrial matrix preproteins contain N-terminal targeting sequences that form the short amphipathic helices (2–6). On the other hand, some mitochondrial proteins of the inner and outer membrane contain internal targeting signals within the mature proteins (7). The mitochondrion has developed a set of delicate translocons to transport the preproteins into the mitochondrial compartments, one translocase of the outer membrane (TOM)² and two translocases of the inner membrane (TIM23 and TIM22) (4, 5, 8). The TOM complex has two surface receptors, Tom20 and Tom70 (9, 10). Tom20 recognizes the N-terminal mitochondrial targeting signals from the preproteins, whereas Tom70 binds to internal targeting sequences of preproteins such as the multi-transmembrane carrier proteins residing in the mitochondrial membranes (9–12). The crystal structure of Saccharomyces cerevisiae Tom70 revealed that Tom70 contained 11 TPR motifs, and the TPR motifs were clustered into two domains. The three TPR motifs in the N-terminal domain of Tom70p form a peptide-binding groove for the C-terminal EEVD motif of Hsp70/Hsp90, whereas the C-terminal domain of Tom70p contains a large preprotein-binding pocket (13).Molecular chaperones Hsp70 and Hsp90 play important roles in targeting the preproteins to TOM complex (14). Hsp70 and Hsp90 can protect these preproteins from aggregation in the cytosol (15). The C-terminal EEVD motifs of Hsp70/Hsp90 may interact directly with the N-terminal domain of Tom70p to target the preproteins to TOM complex (13, 14, 16). The C-terminal EEVD motif of Hsp70/Hsp90 has been indicated to bind several proteins containing TPR motifs including Hop and CHIP. The complex structures for the Hsp70/Hsp90 EEVD motif and Hop and CHIP TPR regions have been determined (17–21).Tom71 (also known as Tom72) was identified as a homologue with Tom70 with high amino acid sequence identity (>50%) (22). Tom71 shares overlapping functions with Tom70 to transfer the preproteins and maintain the mitochondrial morphology (23, 24). In this study, we have determined the crystal structures of S. cerevisiae Tom71 and the complexes of Tom71 and Hsp70/Hsp90 C-terminal EEVD motifs. These structures suggest that the Hsp70/Hsp90 binding to Tom70/Tom71 may keep Tom70/Tom71 in the open state for receiving preproteins. The Hsp70/Hsp90 interactions may also increase the volume of the preprotein-binding pocket of Tom70/Tom71 and prepare Tom70/Tom71 for preprotein loading.     

Biochemical Characterization and Function of Complexes Formed by Hyaluronan and the Heavy Chains of Inter-��-inhibitor (HC��HA) Purified from Extracts of Human Amniotic Membrane

Clinically, amniotic membrane (AM) suppresses inflammation, scarring, and angiogenesis. AM contains abundant hyaluronan (HA) but its function in exerting these therapeutic actions remains unclear. Herein, AM was extracted sequentially with buffers A, B, and C, or separately by phosphate-buffered saline (PBS) alone. Agarose gel electrophoresis showed that high molecular weight (HMW) HA (an average of ∼3000 kDa) was predominantly extracted in isotonic Extract A (70.1 ± 6.0%) and PBS (37.7 ± 3.2%). Western blot analysis of these extracts with hyaluronidase digestion or NaOH treatment revealed that HMW HA was covalently linked with the heavy chains (HCs) of inter-α-inhibitor (IαI) via a NaOH-sensitive bond, likely transferred by the tumor necrosis factor-α stimulated gene-6 protein (TSG-6). This HC·HA complex (nHC·HA) could be purified from Extract PBS by two rounds of CsCl/guanidine HCl ultracentrifugation as well as in vitro reconstituted (rcHC·HA) by mixing HMW HA, serum IαI, and recombinant TSG-6. Consistent with previous reports, Extract PBS suppressed transforming growth factor-β1 promoter activation in corneal fibroblasts and induced mac ro phage apo pto sis. However, these effects were abolished by hyaluronidase digestion or heat treatment. More importantly, the effects were retained in the nHC·HA or rcHC·HA. These data collectively suggest that the HC·HA complex is the active component in AM responsible in part for clinically observed anti-inflammatory and anti-scarring actions.Hyaluronan (HA)⁴ is widely distributed in extracellular matrices, tissues, body fluids, and even in intracellular compartments (reviewed in Refs. 1 and 2). The molecular weight of HA ranges from 200 to 10,000 kDa depending on the source (3), but can also exist as smaller fragments and oligosaccharides under certain physiological or pathological conditions (1). Investigations over the last 15 years have suggested that low M_r HA can induce the gene expression of proinflammatory mediators and proangiogenesis, whereas high molecular weight (HMW) HA inhibits these processes (4–7).Several proteins have been shown to bind to HA (8) such as aggrecan (9), cartilage link protein (10), versican (11), CD44 (12, 13), inter-α-inhibitor (IαI) (14, 15), and tumor necrosis factor-α stimulated gene-6 protein (TSG-6) (16, 17). IαI consists of two heavy chains (HCs) (HC1 and HC2), both of which are linked through ester bonds to a chondroitin sulfate chain that is attached to the light chain, i.e. bikunin. Among all HA-binding proteins, only the HCs of IαI have been clearly demonstrated to be covalently coupled to HA (14, 18). However, TSG-6 has also been reported to form stable, possibly covalent, complexes with HA, either alone (19, 20) or when associated with HC (21).The formation of covalent bonds between HCs and HA is mediated by TSG-6 (22–24) where its expression is often induced by inflammatory mediators such as tumor necrosis factor-α and interleukin-1 (25, 26). TSG-6 is also expressed in inflammatory-like processes, such as ovulation (21, 27, 28) and cervical ripening (29). TSG-6 interacts with both HA (17) and IαI (21, 24, 30–33), and is essential for covalently transferring HCs on to HA (22–24). The TSG-6-mediated formation of the HC·HA complex has been demonstrated to play a crucial role in female fertility in mice. The HC·HA complex is an integral part of an expanded extracellular “cumulus” matrix around the oocyte, which plays a critical role in successful ovulation and fertilization in vivo (22, 34). HC·HA complexes have also been found at sites of inflammation (35–38) where its pro- or anti-inflammatory role remain arguable (39, 40).Immunostaining reveals abundant HA in the avascular stromal matrix of the AM (41, 42).⁵ In ophthalmology, cryopreserved AM has been widely used as a surgical graft for ocular surface reconstruction and exerts clinically observable actions to promote epithelial wound healing and to suppress inflammation, scarring, and angiogenesis (for reviews see Refs. 43–45). However, it is not clear whether HA in AM forms HC·HA complex, and if so whether such an HC·HA complex exerts any of the above therapeutic actions. To address these questions, we extracted AM with buffers of increasing salt concentration. Because HMW HA was found to form the HC·HA complex and was mainly extractable by isotonic solutions, we further purified it from the isotonic AM extract and reconstituted it in vitro from three defined components, i.e. HMW HA, serum IαI, and recombinant TSG-6. Our results showed that the HC·HA complex is an active component in AM responsible for the suppression of TGF-β1 promoter activity, linkable to the scarring process noted before by AM (46–48) and by the AM soluble extract (49), as well as for the promotion of macrophage death, linkable to the inflammatory process noted by AM (50) and the AM soluble extract (51).     

Discovery of keratin function and role in genetic diseases: the year that 1991 was

In 1991, a set of transgenic mouse studies took the fields of cell biology and dermatology by storm in providing the first credible evidence that keratin intermediate filaments play a unique and essential role in the structural and mechanical support in keratinocytes of the epidermis. Moreover, these studies intimated that mutations altering the primary structure and function of keratin filaments underlie genetic diseases typified by cellular fragility. This Retrospective on how these studies came to be is offered as a means to highlight the 25th anniversary of these discoveries.Although intermediate filaments (IFs) have been characterized at some level for a longer period of time (Oshima, 2007 ), they were officially discovered as such as recently as 1968 by Howard Holtzer and colleagues while studying the developing skeletal muscle (Ishikawa et al., 1968 ). The advent of gene cloning methods and monospecific antibody production in the late 1970s and throughout the 1980s led to an explosion of data and knowledge about IFs that established them as a large family of genes and proteins that are individually regulated in a tight and evolutionarily conserved tissue- and differentiation-specific manner. Researchers also uncovered some of the remarkable properties of IFs as purified elements in vitro and in living systems and recognized that they occur in the nucleus as well as in the cytoplasm. In spite of the fast pace of progress during that period, however, it was not possible to produce evidence that spoke unequivocally about the functional importance of IFs in cells and tissues, let alone their role in disease.Beginning in the mid- to late 1980s, pioneering experimentation along two distinct lines was underway in the laboratory of Elaine Fuchs, then at the University of Chicago. The eventual merger of these approaches yielded the first formal insight into IF function in vivo, as well as into their direct involvement in human disease. In an effort to define structure–function relationships with regard to the assembly and network formation properties of IFs, one such approach was the application of systematic deletion mutagenesis to keratin 14 (K14), a type I IF that is expressed with its type II partner keratin 5 (K5) in the progenitor basal layer of the epidermis and related complex epithelia. These studies demonstrated that deleting sequences from either end of the central α-helical rod domain of the K14 protein was deleterious for filament formation in a dominant manner both in transfected cells (Albers and Fuchs, 1987 , 1989 ; Figure 1) and the setting of IF polymerization assays involving purified proteins in vitro (e.g., Coulombe et al., 1990 ). The second key effort in the Fuchs lab in the late 1980s resulted in the demonstration that the proximal 2.5 kb and distal 700 base pairs corresponding respectively to the 5′ upstream and 3′ downstream regions of the cloned human K14 gene were sufficient to confer tissue-specific, that is, K14-like, regulation in transgenic mice in vivo (Vassar et al., 1989 ; Figure 1). This tour de force paved the way for the production of a human K14 gene promoter–based cassette (e.g., Saitou et al., 1995 ) that could reliably direct the expression of any open reading frame in a K14-like manner in transgenic mice. As an aside, this tool has had a profound effect on epithelial and skin biology research.Open in a separate windowFIGURE 1:Schematic representation of the strategy and outcome of the experiments that led to the discovery of keratin function and role in genetic disease. Original figures are reproduced to give a realistic account of the data. (A) Examples of a disrupted keratin filament network in cultured epithelial cells transfected with and expressing a dominantly acting K14 deletion mutant (arrows). (Reproduced from Albers and Fuchs, 1987 , with permission.) (B) Preferential expression of a substance P-epitope–tagged transgenic human K14 protein in the basal layer of tail skin epidermis in mouse, conveying the tissue- and differentiation-specific behavior of the transgene. (Reproduced from Vassar et al., 1989 , with permission.) (C) The two experimental approaches described in A and B were combined to assess the consequences of tissue-specific expression of dominantly acting K14 mutants in skin tissue in vivo. (D) Newborn mouse littermates. The mouse at the top is transgenic (Tg) and expresses a mutated form of K14 in the epidermis. It is showing severe skin blistering (arrows), particularly in its front paws, which are heavily used by mouse newborns to feed from their mother. The bottom mouse is a nontransgenic control showing no such blistering. (E, F) Hematoxylin-eosin–stained skin tissue sections showing the location of subepidermal cleavage within the epidermis of a K14 mutant–expressing transgenic mouse (opposing arrows in E). Cleavage occurs at the level of the basal layer, where the mutant keratin is expressed. Again, this is never seen in control wild-type (Wt) skin (F). Bar, 100 μm (E, F). (D–F are from Coulombe et al., 1991b , with permission.) (G) Leg skin in a patient suffering from the Dowling–Meara form of epidermolysis bullosa simplex. Characteristic of this severe variant of this disease, several skin blisters are often grouped in a herpetiform manner (Fine et al., 1991 ).Subsequent use of the human K14 promoter–based cassette to direct the expression of epitope-tagged and selected deletion mutants of K14 gave rise to transgenic mouse pups that exhibited extensive blistering of the skin preferentially at sites of frictional trauma (Coulombe et al., 1991b ; Vassar et al., 1991 ; Figure 1). Electron microscopy showed that skin blistering occurred secondary to a loss of the integrity of keratinocytes located in the basal layer of the epidermis, that is, the precise site of mutant K14 protein accumulation. Such blistering did not occur in transgenic mice expressing a full-length version of human K14 modified to carry only an epitope tag at the C-terminus at similar or higher levels (Coulombe et al., 1991b ; Vassar et al., 1991 ). In addition, the severity of skin blistering in mutant K14–expressing transgenic pups could be directly related to the extent to which the mutant protein had been shown to disrupt filament assembly in transfected cell assays and in IF reconstitution assays in vitro. For instance, tissue-specific expression of a K14 mutant that could severely disrupt 10-nm filament assembly was associated with whole-body skin blistering and the untimely death of mouse pups and, from a pathology perspective, with “tonofilament clumping” and a paucity of visible keratin IFs in transgenic basal keratinocytes. By comparison, expression of another K14 mutant with a less deleterious effect on 10-nm IF assembly was compatible with the survival of transgenic mouse pups and resulted in skin blistering largely limited to the front paws in newborn mice together with altered organization of keratin IFs in basal keratinocytes of transgenic epidermis in situ, albeit without tonofilament clumping. This initial set of mouse strains thus revealed the existence of a direct link between the so-called “genotype” (i.e., mutant K14 characteristics) and the skin phenotype (Coulombe et al., 1991b ; Vassar et al., 1991 ; Fuchs and Coulombe, 1992 ). Electrophoretic analyses of protein samples confirmed that the K14 mutant proteins acted dominantly to produce such spectacular phenotypes in transgenic mouse skin. Finally, blistering also occurred in the mutant K14–expressing transgenic mice in other stratified epithelia known both to express K14 and experience trauma, notably in the oral mucosa (Coulombe et al., 1991b ; Vassar et al., 1991 ).It is worth celebrating the 25th anniversary of these pioneering experiments for the following two reasons. First, the study of these mice provided the first formal demonstration that keratin IFs play a fundamentally important role in structural support in surface epithelia such as the epidermis and oral mucosa. Without proper IF support, epidermal keratinocytes are rendered fragile and cannot sustain trivial frictional stress (Coulombe et al., 1991b ; Fuchs and Coulombe, 1992 ). The second reason is the observation that the phenotype of these K14 mutant–expressing mice proved eerily similar to those of individuals afflicted with the disease epidermolysis bullosa simplex (EBS), a rare, dominantly inherited and debilitating skin condition in which the epidermis and oral mucosa undergo blistering after exposure to trivial mechanical trauma. As observed in the mouse model, tissue cleavage had been shown to result from the loss of integrity of keratinocytes located in the basal layer (Fine et al., 1991 ). Further, other researchers had previously reported on anomalies in the organization of keratin IFs in the basal epidermal keratinocytes of EBS patients (Anton-Lamprecht, 1983 ; Ito et al., 1991 ) or in cultures of epidermal keratinocytes established from EBS patients (Kitajima et al., 1989 ). The Fuchs laboratory thus teamed up with Amy Paller, a physician-scientist and pediatric dermatologist with deep expertise in genodermatoses, and mutations were soon discovered in the K14 gene of two independent and sporadic cases of a severe variant of the disease known as Dowling–Meara EBS (Coulombe et al., 1991a ; Figure 1). The two mutations were heterozygous missense alleles that affected the very same codon in K14 (Arg-125) and were correctly predicted at the time to correspond to a mutational hot spot in type I keratin genes. The mutations were shown to dominantly disrupt 10-nm IF assembly in vitro and/or in transfected keratinocytes in culture (Coulombe et al., 1991a ). Soon thereafter, a team led by Ervin Epstein at University of California, San Francisco (San Francisco, CA), reported on the use of classical linkage analysis to uncover a missense mutation in the K14 gene of a small pedigree with Koebner-type EBS, a less severe variant of the disease (Bonifas et al., 1991 ). The next year, Birgit Lane and colleagues (Lane et al., 1992 ) reported on the occurrence of mutations in keratin 5 (K5), the formal type II keratin assembly partner for K14 in vivo, in another instance of Dowling–Meara EBS.In the years since 1991, a role in structural support has been formally demonstrated for all classes of IFs (Coulombe et al., 2009 ), including the nuclear-localized lamins (e.g., Lammerding et al., 2004 ). Moreover, we now know of several hundred independent instances of mutations in either K5 or K14 in the setting of the EBS disease, with the vast majority of those consisting of dominantly acting missense alleles (Szeverenyi et al., 2008 ; Human Intermediate Filament Database, www.interfil.org, maintained at the Centre for Molecular Medicine and Bioinformatics Institute, Singapore). We also learned that, as anticipated, EBS largely represents a loss-of-function phenotype, since K14-null mice (Lloyd et al., 1995 ), K14-null individuals (Chan et al., 1994 ; Rugg et al., 1994 ), and K5-null mice (Peters et al., 2001 ) all exhibit an EBS-like skin-blistering phenotype (Coulombe et al., 2009 ). Mutations such as Arg125Cys in K14 markedly compromise the remarkable mechanical properties of keratin filaments (Ma et al., 2001 ), as well as the steady-state dynamics of keratin filaments in transfected keratinocytes in culture (Werner et al., 2004 ). Finally, mutations affecting the coding sequence of IF genes have been shown to underlie >100 diseases affecting the human population (Omary et al., 2004 ; Szeverenyi et al., 2008 ; www.interfil.org). Consistent with the exquisite tissue- and cell type–specific regulation of IF genes, these diseases collectively affect a myriad of tissues and organs and are relevant to nearly all branches of medicine. These observations attest to the importance and profound effect that the generation and characterization of mutant K14–expressing transgenic mice has had for cell biology, epithelial physiology, dermatology, and medicine.Many thoughts spring to mind when reminiscing about my involvement with this body of work. First, this effort was prescient of the power of team science and, in particular, of the potential effect of close collaborations involving biologists and physician-scientists. I learned a great deal and benefited immensely from working closely with many colleagues on this project, including Bob Vassar, Kathryn Albers, Linda Degenstein, Liz Hutton, Anthony Letai, Amy Paller, and, last but not least, my postdoctoral mentor and the laboratory head, Elaine Fuchs. Second, there is no substitute for elements such as innovation, hard work, perseverance, boldness, accountability, and great leadership. Elaine had the vision and created the exceptional circumstances necessary to make this set of discoveries possible, and, of equal importance, she was an integral part of the day-to-day progress and maturation of the entire project. Finally, as we all know, there is an intangible element of luck involved in discovery research. In this instance, a strong argument can be made that the studies highlighted here may not have had such a deep and defining effect had the effort been devoted to any IF other than the K5–K14 keratin pairing.What are some of the lingering issues regarding this specific topic that preoccupy us still, 25 years later? Two challenges loom particularly large. First, we have yet to achieve a satisfactory understanding of how mutations in keratin proteins can cause disease. This is due in part to the lack of an atomic-level understanding of the core structure of IFs (which has been a tough nut to crack; Lee et al., 2012 ), along with the reality that, for any relevant IF gene, there is a broad variety of disease-associated (mostly missense) mutations that pepper their primary structure (www.interfil.org). Second, we have yet to achieve success toward the treatment of EBS or any IF-based disorder. Disease characteristics such as low incidence, a dominantly inherited character, genetic heterogeneity (e.g., broad mutational landscape), and, in the case of EBS and related conditions, an intrinsically high rate of cell turnover within the main target tissue significantly add to the challenge of devising safe and effective therapeutic strategies (Coulombe et al., 2009 ). Although efforts are still underway to foster progress on these two challenging issues, the field as a whole has made significant progress in uncovering a plethora of noncanonical functions of keratin IFs (Hobbs et al., 2016 ) in addition to understanding their regulation, dynamics, and many remarkable properties.     

C-Nap1, a Novel Centrosomal Coiled-Coil Protein and Candidate Substrate of the Cell Cycle–regulated Protein Kinase Nek2

Nek2 (for NIMA-related kinase 2) is a mammalian cell cycle–regulated kinase structurally related to the mitotic regulator NIMA of Aspergillus nidulans. In human cells, Nek2 associates with centrosomes, and overexpression of active Nek2 has drastic consequences for centrosome structure. Here, we describe the molecular characterization of a novel human centrosomal protein, C-Nap1 (for centrosomal Nek2-associated protein 1), first identified as a Nek2-interacting protein in a yeast two-hybrid screen. Antibodies raised against recombinant C-Nap1 produced strong labeling of centrosomes by immunofluorescence, and immunoelectron microscopy revealed that C-Nap1 is associated specifically with the proximal ends of both mother and daughter centrioles. On Western blots, anti–C-Nap1 antibodies recognized a large protein (>250 kD) that was highly enriched in centrosome preparations. Sequencing of overlapping cDNAs showed that C-Nap1 has a calculated molecular mass of 281 kD and comprises extended domains of predicted coiled-coil structure. Whereas C-Nap1 was concentrated at centrosomes in all interphase cells, immunoreactivity at mitotic spindle poles was strongly diminished. Finally, the COOH-terminal domain of C-Nap1 could readily be phosphorylated by Nek2 in vitro, as well as after coexpression of the two proteins in vivo. Based on these findings, we propose a model implicating both Nek2 and C-Nap1 in the regulation of centriole–centriole cohesion during the cell cycle.The serine/threonine kinase NIMA of Aspergillus nidulans is considered the founding member of a family of protein kinases with a possible role in cell cycle regulation (for reviews see Fry and Nigg, 1995; Lu and Hunter, 1995a ; Osmani and Ye, 1996). In A. nidulans, NIMA clearly cooperates with the Cdc2 protein kinase to promote progression into mitosis (Osmani et al., 1991), and overexpression of NIMA in a variety of heterologous species promotes a premature onset of chromosome condensation (O''Connell et al., 1994; Lu and Hunter, 1995b ). This has been interpreted to suggest evolutionary conservation of a pathway involving NIMA-related kinases (for review see Lu and Hunter, 1995a ). Indeed, kinases structurally related to NIMA are present in many species (Fry and Nigg, 1997). However, the only bona fide functional homologue of NIMA so far isolated stems from another filamentous fungus, Neurospora crassa (Pu et al., 1995), and the functional relationship between vertebrate NIMA-related kinases and fungal NIMA remains uncertain.The closest known mammalian relative to NIMA is a kinase termed Nek2 (for NIMA-related kinase 2)¹ (Fry and Nigg, 1997). This kinase undergoes cell cycle–dependent changes in abundance and activity, reminiscent of NIMA (Schultz et al., 1994; Fry et al., 1995). It is highly expressed in male germ cells (Rhee and Wolgemuth, 1997; Tanaka et al., 1997), and data have been reported consistent with a role for Nek2 in meiotic chromosome condensation (Rhee and Wolgemuth, 1997). However, overexpression of active Nek2 in somatic cells has no obvious effect on chromosome condensation; instead, it induces striking alterations in the structure of the centrosome, the principal microtubule-organizing center of mammalian cells (Fry et al., 1998). Furthermore, immunofluorescence microscopy and subcellular fractionation concur to demonstrate that endogenous Nek2 associates with centrosomes, strongly suggesting that one physiological function of this kinase may relate to the centrosome cycle (Fry et al., 1998).The mammalian centrosome is an organelle of about 1 μm in diameter. It comprises two barrel-shaped centrioles that are made of nine short triplet microtubules and are surrounded by an amorphous matrix known as the pericentriolar material (PCM) (for review see Brinkley, 1985; Vorobjev and Nadehzdina, 1987; Kimble and Kuriyama, 1992; Kalt and Schliwa, 1993; Kellogg et al., 1994; Lange and Gull, 1996). Major progress has recently been made with the demonstration that microtubules are nucleated from γ-tubulin–containing ring complexes (γ-TuRCs), which are concentrated within the PCM (Moritz et al., 1995; Zheng et al., 1995). γ-Tubulin forms complexes with Spc97/98, two evolutionarily conserved proteins first identified in budding yeast spindle pole bodies (Geissler et al., 1996; Knop et al., 1997; Stearns and Winey, 1997), and there is also evidence for an important role of pericentrin and other coiled-coil proteins in organizing γ-TuRCs into higher order lattice structures (Doxsey et al., 1994; Dictenberg et al., 1998). However, in spite of this recent progress, it is clear that the inventory of centrosome components is far from complete.Centrosome structure and function is regulated in a cell cycle–dependent manner (for reviews see Mazia, 1987; Kellogg et al., 1994; Tournier and Bornens, 1994). Once in every cell cycle, and beginning around the G1/S transition, centrioles are duplicated (e.g., Kuriyama and Borisy, 1981a ; Vorobjev and Chentsov, 1982; Kochanski and Borisy, 1990; Chrétien et al., 1997). Late in G2, centrosomes then grow in size (a process referred to as maturation) through the recruitment of additional PCM proteins (Rieder and Borisy, 1982; Kalt and Schliwa, 1993; Lange and Gull, 1995). At the G2/M transition, the duplicated centrosomes separate and migrate to opposite ends of the nucleus. Concomitantly, their microtubule-nucleating activities increase dramatically in preparation for spindle formation (McGill and Brinkley, 1975; Snyder and McIntosh, 1975; Gould and Borisy, 1977; Kuriyama and Borisy, 1981b ; for reviews see Brinkley, 1985; Vorobjev and Nadehzdina, 1987; Karsenti, 1991). By what mechanisms these events are controlled remains largely unknown, but data obtained using phosphoepitope-specific antibodies strongly suggest that phosphorylation of centrosomal proteins plays a major role (Vandré et al., 1984, 1986; Centonze and Borisy, 1990). More direct support for this view stems from the observation that cyclin-dependent kinases (CDKs) enhance the microtubule-nucleation activity of centrosomes at the G2/M transition (Verde et al., 1990, 1992; Buendia et al., 1992) and are involved in promoting centrosome separation (Blangy et al., 1995; Sawin and Mitchison, 1995). Similarly, polo-like kinase 1, a cell cycle regulatory kinase structurally distinct from CDKs, has recently been implicated in centrosome maturation (Lane and Nigg, 1996).The precise role of Nek2 at the centrosome remains to be determined, but it is intriguing that overexpression of this kinase in human cells causes a pronounced splitting of centrosomes. This led us to propose that Nek2-dependent phosphorylation of previously unidentified proteins may cause a loss of centriole–centriole cohesion, and that this event might represent an early step in centrosome separation at the G2/M transition (Fry et al., 1998). With the aim of identifying potential substrates (or regulators) of Nek2, we have now performed a yeast two-hybrid screen, using full-length Nek2 as a bait. We report here the molecular characterization of a novel coiled-coil protein that we call C-Nap1 (for centrosomal Nek2-associated protein 1). C-Nap1 represents a core component of the mammalian centrosome and the first candidate substrate for a member of the NIMA protein kinase family to be identified.     

Genetic Analysis of the nuo Locus,Which Encodes the Proton-Translocating NADH Dehydrogenase in Escherichia coli

Complex I (EC 1.6.99.3) of the bacterium Escherichia coli is considered to be the minimal form of the type I NADH dehydrogenase, the first enzyme complex in the respiratory chain. Because of its small size and relative simplicity, the E. coli enzyme has become a model used to identify and characterize the mechanism(s) by which cells regulate the synthesis and assembly of this large respiratory complex. To begin dissecting the processes by which E. coli cells regulate the expression of nuo and the assembly of complex I, we undertook a genetic analysis of the nuo locus, which encodes the 14 Nuo subunits comprising E. coli complex I. Here we present the results of studies, performed on an isogenic collection of nuo mutants, that focus on the physiological, biochemical, and molecular consequences caused by the lack of or defects in several Nuo subunits. In particular, we present evidence that NuoG, a peripheral subunit, is essential for complex I function and that it plays a role in the regulation of nuo expression and/or the assembly of complex I.

Complex I (NADH:ubiquinone oxidoreductase; EC 1.6.99.3), a type I NADH dehydrogenase that couples the oxidation of NADH to the generation of a proton motive force, is the first enzyme complex of the respiratory chain (2, 35, 47). The Escherichia coli enzyme, considered to be the minimal form of complex I, consists of 14 subunits instead of the 40 to 50 subunits associated with the homologous eukaryotic mitochondrial enzyme (17, 29, 30, 48–50). E. coli also possesses a second NADH dehydrogenase, NDH-II, which does not generate a proton motive force (31). E. coli complex I resembles eukaryotic complex I in many ways (16, 17, 30, 49): it performs the same enzymatic reaction and is sensitive to a number of the same inhibitors, it consists of subunits homologous to those found in all proton-translocating NADH:ubiquinone oxidoreductases studied thus far, it is comprised of a large number of subunits relative to the number that comprise other respiratory enzymes, and it contains flavin mononucleotide and FeS center prosthetic groups. Additionally, it possesses an L-shaped topology (14, 22) like that of its Neurospora crassa homolog (27), and it consists of distinct fragments or subcomplexes. Whereas eukaryotic complex I can be dissected into a peripheral arm and a membrane arm, the E. coli enzyme consists of three subcomplexes referred to as the peripheral, connecting, and membrane fragments (29) (Fig. ​(Fig.1A).1A). The subunit composition of these three fragments correlates approximately with the organization of the 14 structural genes (nuoA to nuoN) (49) of the nuo (for NADH:ubiquinone oxidoreductase) locus (Fig. ​(Fig.1B),1B), an organization that is conserved in several other bacteria, including Salmonella typhimurium (3), Paracoccus denitrificans (53), Rhodobacter capsulatus (12), and Thermus thermophilus (54). The 5′ half of the locus contains a promoter (nuoP), previously identified and located upstream of nuoA (8, 49), and the majority of genes that encode subunits homologous to the nucleus-encoded subunits of eukaryotic complex I and to subunits of the Alcaligenes eutrophus NAD-reducing hydrogenase (17, 29, 30, 49). In contrast, the 3′ half contains the majority of the genes that encode subunits homologous to the mitochondrion-encoded subunits of eukaryotic complex I and to subunits of the E. coli formate-hydrogen lyase complex (17, 29, 30, 49). Whereas the nuclear homologs NuoE, NuoF, and NuoG constitute the peripheral fragment (also referred to as the NADH dehydrogenase fragment [NDF]), the nuclear homologs NuoB, NuoC, NuoD, and NuoI constitute the connecting fragment. The mitochondrial homologs NuoA, NuoH, NuoJ, NuoK, NuoL, NuoM, and NuoN constitute the membrane fragment (29). E. coli complex I likely evolved by fusion of preexisting protein assemblies constituting modules for electron transfer and proton translocation (17–19, 30). Open in a separate windowFIG. 1Schematic of E. coli complex I and the nuo locus. Adapted with permission of the publisher (17, 29, 30, 49). (A) E. coli complex I is comprised of three distinct fragments: the peripheral (light gray), connecting (white), and membrane (dark gray) fragments (17, 29). The peripheral fragment (NDF) is comprised of the nuclear homologs NuoE, -F, and -G and exhibits NADH dehydrogenase activity that oxidizes NADH to NAD⁺; the connecting fragment is comprised of the nuclear homologs NuoB, -C, -D, and -I; and the membrane fragment is comprised of the mitochondrial homologs NuoA, -H, and -J to -N and catalyzes ubiquinone (Q) to its reduced form (QH₂). FMN, flavin mononucleotide. (B) The E. coli nuo locus encodes the 14 Nuo subunits that constitute complex I. The 5′ half of the locus contains a previously identified promoter (nuoP) and the majority of genes that encode the peripheral and connecting subunits (light gray and white, respectively). The 3′ half of the locus contains the majority of the genes encoding the membrane subunits (dark gray). The 3′ end of nuoG encodes a C-Terminal region (CTR) of the NuoG subunit (hatched).Because of its smaller size and relative simplicity, researchers recently have begun to utilize complex I of E. coli, and that of its close relative S. typhimurium, to identify and characterize the mechanism(s) by which cells regulate the synthesis and assembly of this large respiratory complex (3, 8, 46) and to investigate the diverse physiological consequences caused by defects in this enzyme (4, 6, 10, 40, 59). Such defects affect the ability of cells to perform chemotaxis (40), to grow on certain carbon sources (4, 6, 10, 40, 57), to survive stationary phase (59), to perform energy-dependent proteolysis (4), to regulate the expression of at least one gene (32), and to maintain virulence (5).To begin dissecting the processes by which E. coli cells regulate the expression of nuo and the assembly of complex I, we undertook a genetic analysis of the nuo locus. Here, we present the results of studies, performed on an isogenic collection of nuo mutants, that focus on the physiological, biochemical, and molecular consequences caused by the lack of or defects in several Nuo subunits. In particular, we present evidence that NuoG, a peripheral subunit, is essential for complex I function and that it plays a role in the regulation of nuo expression and/or the assembly of complex I.