Differential polyubiquitin recognition by tandem ubiquitin binding domains of Rabex-5

The biphenyl dioxygenase of Burkholderia xenovorans LB400 (BphAE(LB400)) is a Rieske-type oxygenase that catalyzes the stereospecific oxygenation of many heterocyclic aromatics including dibenzofuran. In a previous work, we evolved BphAE(LB400) and obtained BphAE(RR41). This variant metabolizes dibenzofuran and 2-chlorodibenzofuran more efficiently than BphAE(LB400). However, the regiospecificity of BphAE(RR41) toward these substrates differs. Dibenzofuran is metabolized principally through a lateral dioxygenation whereas 2-chlorodibenzofuran is metabolized principally through an angular dioxygenation. In order to explain this difference, we examined the crystal structures of both substrate-bound forms of BphAE(RR41) obtained under anaerobic conditions. This structure analysis, in combination with biochemical data for a Ser283Gly mutant provided evidences that the substrate is compelled to move after oxygen-binding in BphAE(RR41):dibenzofuran. In BphAE(RR41):2-chlorodibenzofuran, the chlorine atom is close to the side chain of Ser283. This contact is missing in the BphAE(RR41):dibenzofuran, and strong enough in the BphAE(RR41):2-chlorodibenzofuran to help prevent substrate movement during the catalytic reaction.     

A ubiquitin C-terminal isopeptidase that acts on polyubiquitin chains. Role in protein degradation.

In the ubiquitin (Ub) pathway, proteins are ligated with polyUb chains and then are degraded by a 26 S protease complex. We describe an enzyme, called isopeptidase T, that acts on polyUb chains. It is a monomeric Ub-binding protein abundant in erythrocytes and reticulocytes. The activity of the isopeptidase is inhibited by iodoacetamide and Ub aldehyde. Treatment of the enzyme with Ub aldehyde increased its affinity for free Ub, indicating the existence of two different Ub-binding sites and cooperativity between the two sites. Isopeptidase T acts on polyUb-protein conjugates, but not on conjugates in which the formation of polyUb chains was prevented by the use of reductively methylated Ub or on abnormal polyUb chains formed with a mutant Ub that contains a Lys----Arg substitution at residue 48. The enzyme converts high molecular mass polyUb-protein conjugates to lower molecular mass forms with the release of free Ub, but not of free protein substrate. The lower molecular mass Ub-protein conjugate products are resistant to further action of the enzyme. Isopeptidase T stimulates protein degradation in a system reconstituted from purified enzyme components. The enzyme also stimulates the degradation of proteins ligated to polyUb chains by the 26 S protease complex. Preincubation of polyUb-protein conjugates with the isopeptidase did not much increase their susceptibility to proteolysis by the 26 S complex. On the other hand, preincubation of conjugates with the 26 S protease complex and ATP increased the release of free Ub upon further incubation with the isopeptidase. It thus seems that a role of this isopeptidase in protein breakdown is to remove polyUb chain remnants following the degradation of the protein substrate moiety by the 26 S complex.     

Characterization of ubiquitin and ubiquitin-like-protein isopeptidase activities

Conjugation or deconjugation of ubiquitin (Ub) or ubiquitin-like proteins (UBLs) to or from cellular proteins is a multifaceted and universal means of regulating cellular physiology, controlling the lifetime, localization, and activity of many critical proteins. Deconjugation of Ub or UBL from proteins is performed by a class of proteases called isopeptidases. Herein is described a readily quantifiable novel isopeptidase assay platform consisting of Ub or UBL fused to the reporter enzyme phospholipase A(2) (PLA(2)). Isopeptidase activity releases PLA(2), which cleaves its substrate, generating a signal that is linear with deubiquitylase (DUB) concentration and is able to discriminate DUB, deSUMOylase, deNEDDylase, and deISGylase activities. The power and sensitivity of the UBL-PLA(2) assay are demonstrated by its ability to differentiate the contrasting deISGylase and DUB activities of two coronavirus proteases: severe acute respiratory syndrome papain-like protease (SARS-CoV PLpro) and NL63 CoV papain-like protease 2 (PLP2). Furthermore, direct comparisons with the current Ub-7-amino-4-methylcoumarin (Ub-AMC) assay demonstrated that the Ub-PLA(2) assay is an effective tool for characterizing modulators of isopeptidase activity. This observation was expanded by profiling the inhibitory activity of the nonselective isopeptidase inhibitor NSC 632839 against DUBs and deSUMOylases. Taken together, these studies illustrate the utility of the reporter-based approach to measuring isopeptidase activity.     

Analysis of ubiquitin E3 ligase activity using selective polyubiquitin binding proteins

The ubiquitin proteasome pathway controls the cellular degradation of ~80-90% of the proteome in a highly regulated manner. In this pathway, E3 ligases are responsible for the conjugation of ubiquitin to protein substrates which can lead to their destruction by the 26S proteasome. Aberrant E3 ligases have been implicated in several diseases and are widely recognized as attractive targets for drug discovery. As researchers continue to characterize E3 ligases, additional associations with various disease states are being exposed. The availability of assays that allow rapid analysis of E3 ligase activity is paramount to both biochemical studies and drug discovery efforts aimed at E3 ligases. To address this need, we have developed a homogenous assay for monitoring ubiquitin chain formation using Tandem Ubiquitin Binding Entities (TUBEs). TUBEs bind selectively to polyubiquitin chains versus mono-ubiquitin thus enabling the detection of polyubiquitin chains in the presence of mono-ubiquitin. This assay reports on the proximity between the protein substrate and TUBEs as a result of polyubiquitin chain formation by an E3 ligase. This homogenous assay is a step forward in streamlining an approach for characterizing and quantitating E3 ligase activity in a rapid and cost effective manner. This article is part of a Special Issue entitled: Ubiquitin Drug Discovery and Diagnostics.     

Recognition of the polyubiquitin proteolytic signal

Polyubiquitin chains linked through Lys48 are the principal signal for targeting substrates to the 26S proteasome. Through studies of structurally defined, polyubiquitylated model substrates, we show that tetraubiquitin is the minimum signal for efficient proteasomal targeting. The mechanism of targeting involves a simple increase in substrate affinity that is brought about by autonomous binding of the polyubiquitin chain. Assigning the proteasomal signaling function to a specific polymeric unit explains how a single ubiquitin can act as a functionally distinct signal, for example in endocytosis. The properties of the substrates studied here implicate substrate unfolding as a kinetically dominant step in the proteolysis of properly folded proteins, and suggest that extraproteasomal chaperones are required for efficient degradation of certain proteasome substrates.     

Molecular determinants of polyubiquitin linkage selection by an HECT ubiquitin ligase

Ubiquitin (Ub)-protein ligases (E3s) frequently modify their substrates with multiple Ub molecules in the form of a polyubiquitin (poly-Ub) chain. Although structurally distinct poly-Ub chains (linked through different Ub lysine (Lys) residues) can confer different fates on target proteins, little is known about how E3s select the Lys residue to be used in chain synthesis. Here, we used a combination of mutagenesis, biochemistry, and mass spectrometry to map determinants of linkage choice in chain assembly catalyzed by KIAA10, an HECT (Homologous to E6AP C-Terminus) domain E3 that synthesizes K29- and K48-linked chains. Focusing on the Ub molecule that contributes the Lys residue for chain formation, we found that specific surface residues adjacent to K48 and K29 are critical for the usage of the respective Lys residues in chain synthesis. This direct mechanism of linkage choice bears similarities to the mechanism of substrate site selection in sumoylation catalyzed by Ubc9, but is distinct from the mechanism of chain linkage selection used by the Mms2/Ubc13 (Ub E2 variant (UEV)/E2) complex.     

Cyclization of polyubiquitin by the E2-25K ubiquitin conjugating enzyme

For most substrates of ubiquitin (Ub)-dependent degradation, recognition by the proteasome is mediated by a covalently attached signal assembled from multiple ubiquitins linked to each other via the C terminus of one Ub and the epsilon-amine of Lys(48) of another Ub. Among Ub-conjugating enzymes, E2-25K is unique in its ability to synthesize in vitro unanchored Lys(48)-linked poly-Ub chains from mono- or poly-Ub, E1, and ATP; thus, E2-25K has distinct binding sites for donor and acceptor (poly)Ub. During studies of chain assembly by E2-25K, we observed that Lys(48)-linked tri-Ub was efficiently converted to a new species that upon SDS-polyacrylamide gel electrophoresis migrated between linear di-Ub and tri-Ub. Analysis of this product by mass spectrometry and tryptic digestion showed that it was a cyclic form of tri-Ub. Cyclization of tri-Ub requires E1, E2-25K, ATP, and that the linear substrate has a free Gly(76) C terminus on the proximal end Ub and a Lys(48) side chain available on the distal end Ub. E2-25K similarly can catalyze the cyclization of longer poly-Ub chains, including tetra- and penta-Ub. Although cyclic tri-Ub resists hydrolysis by the PA700 or isopeptidase T deubiquitinating enzymes, it can be disassembled to Ub monomers by isopeptidase(s) in a red blood cell extract. Thus, if cyclic poly-Ub forms in vivo, it will not accumulate as a dead-end product.     

Regulation of polyubiquitin genes to meet cellular ubiquitin requirement

Ubiquitin (Ub) is one of the proteins that are highly conserved from yeast to humans. It is an essential core unit of the well-defined post-translational modification, called ubiquitination, which is involved in a variety of biological processes. In meta-zoans, Ub is encoded by two monoubiquitin genes and two polyubiquitin genes, in which a single Ub is fused to a ribosomal protein or Ub coding units are arranged in tandem repeats. In mice, polyubiquitin genes (Ubb and Ubc) play a pivotal role to meet the requirement of cellular Ub pools during embryonic development. In addition, expression levels of polyubiquitin genes are increased to adapt to environmental stimuli such as oxidative, heat-shock, and proteotoxic stress. Several researchers have reported about the perturbation of Ub pools through genetic alteration or exogenous Ub delivery using diverse model systems. To study Ub pool changes in a physiologically relevant manner, changing Ub pools via the regulation of endogenous polyubiquitin gene expression has recently been introduced. Furthermore, to understand the regulation of polyubiquitin gene expression more precisely, cis-acting elements and trans-acting factors, which are regulatory components of polyubiquitin genes, have been analyzed. In this review, we discuss how the role of polyu-biquitin genes has been studied during the past decade, es-pecially focusing on their regulation.     

Sequestosome 1/p62 is a polyubiquitin chain binding protein involved in ubiquitin proteasome degradation

Herein, we demonstrate that the ubiquitin-associated (UBA) domain of sequestosome 1/p62 displays a preference for binding K63-polyubiquitinated substrates. Furthermore, the UBA domain of p62 was necessary for aggregate sequestration and cell survival. However, the inhibition of proteasome function compromised survival in cells with aggregates. Mutational analysis of the UBA domain reveals that the conserved hydrophobic patch MGF as well as the conserved leucine in helix 2 are necessary for binding polyubiquitinated proteins and for sequestration-aggregate formation. We report that p62 interacts with the proteasome by pull-down assay, coimmunoprecipitation, and colocalization. Depletion of p62 levels results in an inhibition of ubiquitin proteasome-mediated degradation and an accumulation of ubiquitinated proteins. Altogether, our results support the hypothesis that p62 may act as a critical ubiquitin chain-targeting factor that shuttles substrates for proteasomal degradation.     

Activation of the endosome-associated ubiquitin isopeptidase AMSH by STAM, a component of the multivesicular body-sorting machinery

AMSH is an endosomal ubiquitin isopeptidase that can limit EGF receptor downregulation . It directly binds to the SH3 domain of STAM, which is constitutively associated with Hrs, a component of clathrin-coated structures on endosomes. This clathrin coat has been implicated in the recruitment of ubiquitinated growth factor receptors prior to their incorporation into internal vesicles of the multivesicular body (MVB) , through the concerted action of ESCRT complexes I, II, and III . We now show that AMSH is embedded within a network of interactions with components of the MVB-sorting machinery. AMSH and STAM, like Hrs , both bind directly to clathrin. AMSH also interacts with mVps24/CHMP3, a component of ESCRT III complex, and this interaction is reinforced through simultaneous STAM binding. We have explored the effect of interacting components on the in vitro enzymatic activity of AMSH. The enzyme shows specificity for K63- over K48-linked polyubiquitin chains in vitro and is markedly stimulated by coincubation with STAM, indicating that activation of AMSH is coupled to its association with the MVB-sorting machinery. Other interacting factors do not directly stimulate AMSH but may serve to orient the enzyme with respect to substrates on the endosomal membrane.     

The ISG15 isopeptidase UBP43 is regulated by proteolysis via the SCFSkp2 ubiquitin ligase

The Skp2 oncoprotein belongs to the family of F-box proteins that function as substrate recognition factors for SCF (Skp1, cullin, F-box protein) E3 ubiquitin-ligase complexes. Binding of the substrate to the SCFSkp2 complex catalyzes the conjugation of ubiquitin molecules to the bound substrate, resulting in multi-ubiquitination and rapid degradation by the 26 S proteasome. Using Skp2 as bait in a yeast two-hybrid screen, we have identified UBP43 as a novel substrate for Skp2. UBP43 belongs to the family of ubiquitin isopeptidases and specifically cleaves ISG15, a ubiquitin-like molecule that is induced by cellular stresses, such as type 1 interferons (IFN), nephrotoxic damage, and bacterial infection. UBP43 was originally identified as an up-regulated gene in knock-in mice expressing an acute myelogenous leukemia fusion protein, AML1-ETO, as well as in melanoma cell lines treated with IFN-beta. The phenotype of UBP43 knockout mice includes shortened life span, hypersensitivity to IFN, and neuronal damage, suggesting that tight regulation of ISG15 conjugation is critical for normal cellular function. In this study, we demonstrate that UBP43 is ubiquitinated in vivo and accumulates in cells treated with proteasome inhibitors. We also show that Skp2 promotes UBP43 ubiquitination and degradation, resulting in higher levels of ISG15 conjugates. In Skp2-/- mouse cells, levels of UBP43 are consistently up-regulated, whereas levels of ISG15 conjugates are reduced. Our results demonstrate that the SCFSkp2 is involved in controlling UBP43 protein levels and may therefore play an important role in modulating type 1 IFN signaling.     

Mechanism of polyubiquitin chain recognition by the human ubiquitin conjugating enzyme Ube2g2

Ube2g2 is a human ubiquitin conjugating (E2) enzyme involved in the endoplasmic reticulum-associated degradation pathway, which is responsible for the identification and degradation of unfolded and misfolded proteins in the endoplasmic reticulum compartment. The Ube2g2-specific role is the assembly of Lys-48-linked polyubiquitin chains, which constitutes a signal for proteasomal degradation when attached to a substrate protein. NMR chemical shift perturbation and paramagnetic relaxation enhancement approaches were employed to characterize the binding interaction between Ube2g2 and ubiquitin, Lys-48-linked diubiquitin, and Lys-63-linked diubiquitin. Results demonstrate that ubiquitin binds to Ube2g2 with an affinity of 90 μM in two different orientations that are rotated by 180° in models generated by the RosettaDock modeling suite. The binding of Ube2g2 to Lys-48- and Lys-63-linked diubiquitin is primarily driven by interactions with individual ubiquitin subunits, with a clear preference for the subunit containing the free Lys-48 or Lys-63 side chain (i.e. the distal subunit). This preference is particularly striking in the case of Lys-48-linked diubiquitin, which exhibits an ～3-fold difference in affinities between the two ubiquitin subunits. This difference can be attributed to the partial steric occlusion of the subunit whose Lys-48 side chain is involved in the isopeptide linkage. As such, these results suggest that Lys-48-linked polyubiquitin chains may be designed to bind certain proteins like Ube2g2 such that the terminal ubiquitin subunit carrying the reactive Lys-48 side chain can be positioned properly for chain elongation regardless of chain length.     

Further characterization of the putative human isopeptidase T catalytic site

The human isopeptidase T (isoT) is a zinc-binding deubiquitinating enzyme involved in the disassembly of free K48-linked polyubiquitin chains into ubiquitin monomers. The catalytic site of this enzyme is thought to be composed of Cys335, Asp435, His786 and His795. These four residues were site-directed mutagenized. None of the mutants were able to cleave a peptide-linked ubiquitin dimer. Similarly, C335S, D435N and H795N mutants had virtually no activity against a K48-linked isopeptide ubiquitin dimer, which is an isoT-specific substrate that mimics the K48-linked polyubiquitin chains. On the other hand, the H786N mutant retained a partial activity toward the K48-linked substrate, suggesting that the His786 residue might not be part of the catalytic site. None of the mutations significantly affected the capacity of isoT to bind ubiquitin and zinc. Thus, the catalytic site of UBPs could resemble that of other cysteine proteases, which contain one Cys, one Asp and one His.     

Phylogenetic relationship of ubiquitin repeats in the polyubiquitin gene from the marine sponge Geodia cydonium

Ubiquitin is a 76-residue protein which is highly conserved among eukaryotes. Sponge (Porifera) ubiquitin, isolated from Geodia cydonium, is encoded by a gene (termed GCUBI) with six repeats, GCUBI-1 to GCUBI-6. All repeat units encode the same protein (with one exception: GCUBI-4 encodes ubiquitin with a change of Leu to Val at position 71). On the nt level the sequences of the six repeats differ considerably. All changes (except in GCUBI-4) are silent substitutions, which do not affect the protein structure. However, there is one major difference between the repeats: Codons from both codon families (TCN and AGPy) are simultaneously used for the serine at position 65. Using this characteristic the repeats were divided into two groups: Group I: GCUBI-1,3 (TCT codon) and GCUBI-5 (TCC); Group II: GCUBI-2,4,6 (AGC codon). Mutational analysis suggests that the sponge polyubiquitin gene evolved from an ancestral monoubiquitin gene by gene duplication and successive tandem duplications. The ancestral monoubiquitin gene most probably coded for threonine (ACC) at position 65. The first event, duplication of the monoubiquitin gene, happened some 110 million years ago. Since sponges from the genus Geodia are known from the Cretaceous (145 million) to recent time, it is very likely that all events in the evolution of polyubiquitin gene occurred in the same genus. Alignment data of the consensus ubiquitin nt sequences of different animals (man to protozoa) reflect very well the established phylogenetic relationships of the chosen organisms and show that the sponge ubiquitin gene branched off first from the multicellular organisms.Correspondence to: W.E.G. Müller     

Different HECT domain ubiquitin ligases employ distinct mechanisms of polyubiquitin chain synthesis

Individual ubiquitin (Ub)-protein ligases (E3s) cooperate with specific Ub-conjugating enzymes (E2s) to modify cognate substrates with polyubiquitin chains. E3s belonging to the Really Interesting New Gene (RING) and Homologous to E6-Associated Protein (E6AP) C-Terminus (HECT) domain families utilize distinct molecular mechanisms. In particular, HECT E3s, but not RING E3s, form a thiol ester with Ub before transferring Ub to the substrate lysine. Here we report that different HECT domain E3s can employ distinct mechanisms of polyubiquitin chain synthesis. We show that E6AP builds up a K48-linked chain on its HECT cysteine residue, while KIAA10 builds up K48- and K29-linked chains as free entities. A small region near the N-terminus of the conserved HECT domain helps to bring about this functional distinction. Thus, a given HECT domain can specify both the linkage of a polyubiquitin chain and the mechanism of its assembly.     

Recognition of modified forms of ribonuclease A by the ubiquitin system

The substrate specificity of the ubiquitin (Ub) conjugation system was explored with regard to recognition of unfolded conformation and/or oxidized methionine residues in six derivatives of bovine RNase A. Based on the following observations, ubiquitination of RNase A substrates by the enzymes in a rabbit reticulocyte extract appears to correlate with unfolded conformation rather than with methionine oxidation. 1) Methionine oxidation in already unfolded forms of RNase A does not enhance ubiquitination. 2) Fluorescence measurements and iodoacetate trapping of free sulfhydryls show that the disulfide bonds of MetSO-RNase A, in which the 4 methionine residues are oxidized to the sulfoxide, are reduced by 2 mM dithiothreitol (DTT) in standard Ub conjugation assays so that this derivative also is unfolded. 3) Although MetSO-RNase A is ubiquitinated in the absence of DTT, its intrinsic fluorescence, cation-exchange properties, and susceptibility to reduction indicate a non-native conformation. 4) Methionine sulfoxide-containing peptides that mimic regions of RNase A fail to inhibit conjugation of 125I-Ub to MetSO-RNase A. Ub adducts to two of the six derivatives (MetSO- and reduced/carboxamidomethylated MetSO-RNase A) increase when DTT is omitted from the reactions. Ubaldehyde, an inhibitor of isopeptidases that disassemble Ub-protein conjugates, increased product yields and reduced or abolished the DTT effect, suggesting that an isopeptidase specific for these two RNase A derivatives may be inactivated by oxidation. Ub conjugates of the other RNase A derivatives also increase with Ub-aldehyde but are unaffected by DTT.     

Zinc is required for the catalytic activity of the human deubiquitinating isopeptidase T

Two recombinant human isopeptidase T isoforms, ISOT-S and ISOT-L, differing by an insertion of 23 amino acids in ISOT-L, were previously classified as thiol proteases. Both contain one Zn2+-binding site of high-affinity, which is part of a cryptic nitrilo-triacetate-resistant pocket (site 1). A second Zn2+ site (site 2) was disclosed when both isoforms of the holoenzyme were incubated with an excess of Zn2+. The firmly bound Zn2+ of site 1 could be removed either slowly by dialysis against 1,10-phenanthroline at pH 5.5 or rapidly by treatment at pH 3.0 in the presence of 6 M urea followed by gel filtration at neutral pH. Zn2+ in site 1, but not in site 2, is essential for proteolytic activity because apoproteins were inactive. Inhibition of the catalytic activity was not due to a loss of ubiquitin binding capacity. CD spectra of both isoforms disclosed no major structural differences between the apo- and holoenzymes. The reconstitution of apoenzyme with Zn2+ under nondenaturing conditions at pH 5.5 completely restored enzymatic activity, which was indistinguishable from the reconstitution carried out in urea at pH 3.0. Thus, both human ISOTs are either thiol proteases with a local structural Zn2+ or monozinc metalloproteases that might use in catalysis a Zn2+-activated hydroxide ion polarized by Cys335.     

The missing links to link ubiquitin: Methods for the enzymatic production of polyubiquitin chains

Attachment of ubiquitin (Ub) as monoUb and polyUb chains of different lengths and linkages to proteins plays a dominant role in very different regulatory mechanisms. Therefore, the study of polyUb chains has assumed a central interest in biochemistry and structural biology. An essential step necessary to allow in vitro biochemical and structural studies of polyUbs is the production of their chains in high quantities and purity. This is not always an easy task and can be achieved both enzymatically and chemically. Previous reviews have covered chemical cross-linking exhaustively. In this review, we concentrate on the different approaches developed so far for the enzymatic production of different Ub chains. These strategies permit a certain flexibility in the production of chains with various linkages and lengths. We critically describe the available methods and comment on advantages and limitations. It is clear that the field is mature to study most of the possible links, but some more work needs to be done to complete the picture and to exploit the current methodologies for understanding in full the Ub code.     

Expression of multiple troponin T isoforms in chicken breast muscle regeneration induced by sub-serous implantation

Chicken fast-muscle type (F-type) troponin T (TnT) isoforms are classified into two types, leg-muscle type (L-type) and breast-muscle type (B-type), which are generated by exclusion and inclusion of exon x series-derived sequences in mRNAs, respectively. The B-type isoforms are further classified into neonatal breast-muscle (BN), young chicken breast-muscle (BC), and adult chicken breast-muscle (BA) subtypes. It is known that the multiple F-type TnT isoforms are transiently expressed in the breast muscle tissue during normal development. To examine whether the transition of the isoforms was fixed in muscle cell lineage, breast muscle pieces (pectoralis major) of 1-day old chicks were cultured under gizzard serous membrane of the same chicks for 60 days at the longest. TnT isoform expression of the implants was monitored by immunoblotting and immunostaining using anti-F-type TnT against both L-type and B-type isoforms, anti-exon x3 against only B-type isoforms, and anti-S-type TnT against slow-muscle-type (S-type) isoforms. Muscle fibers in the implant degenerated first, and then new myotubes expressing L-type isoforms were formed by the fusion of myoblasts from surviving satellite cells. When the maturation of the myotubes into myofibers proceeded, BN-, BC-, and BA-subtype isoforms were expressed in the order of developmental stage specific-manner, indicating that the order of appearance of these isoforms was fixed in muscle cell lineage. In immunostaining of the implants recovered on the 60th day after implantation, at least three kinds of the regenerated myofibers were observed, expressing mainly B-type, both B-type and L-type, and only L-type isoforms. The immunohistochemical results suggested that the regulation of alternative splicing of F-type TnT pre-mRNAs was different among individual myofibers, and that the regulation was programmed in myogenic cells, probably satellite cells, which were the primary source of the fibers.