The Acetyltransferase RibT From Bacillus subtilis Affects in vivo Dynamics of the Multimeric Heavy Riboflavin Synthase Complex.

Flavins are ubiquitous molecules in life as they serve as important enzyme cofactors. In the Gram-positive, soil-dwelling bacterium Bacillus subtilis, four well-characterized gene products (the enzymes RibDG, RibE, RibAB, and RibH) catalyze the biosynthesis of riboflavin (RF) from guanosine-triphosphate (GTP) and ribulose-5-phosphate (R5P). The corresponding genes form an operon together with the gene ribT (ribDG-E-AB-H-T), wherein the function of this terminal gene remained enigmatic. RibT has been structurally characterized as a GCN5-like acetyltransferase (GNAT), however, with unidentified target molecules. Bacterial two-hybrid system revealed interactions between RibT, RibH, and RibE, forming the heavy RF synthase complex. Applying single particle tracking (SPT), we found that confined (sub)diffusion of RibT is largely dependent on interacting RibE and, to a lesser degree, on interacting RibH. By induced expression of otherwise low-expressed ribT from an ectopic locus, we observed a decrease in the subpopulation considered to represent capsids of the heavy RF synthase and an increase in the subpopulation thought to represent pentamers of RibH, pointing to a putative role for RibT in capsid disassembly. Complementarily, either deletion of ribT or mutation of a key residue from RibH (K29) suspected to be the substrate of RibT for acetylation leads to increased levels of subpopulations considered as capsids of RibH-mVenus (RibH-mV) in comparison to wild-type (wt)-like cells. Thus, we provide evidence for an indirect involvement of RibT in RF biosynthesis by a putative capsid disassembling mechanism considered to involve acetylation of RibH residue K29 at the three-fold symmetry axis of 60-mer capsids.

Structure of the ubiquitin-activating enzyme loaded with two ubiquitin molecules.

The activation of ubiquitin by the ubiquitin-activating enzyme Uba1 (E1) constitutes the first step in the covalent modification of target proteins with ubiquitin. This activation is a three-step process in which ubiquitin is adenylated at its C-terminal glycine, followed by the covalent attachment of ubiquitin to a catalytic cysteine residue of Uba1 and the subsequent adenylation of a second ubiquitin. Here, a ubiquitin E1 structure loaded with two ubiquitin molecules is presented for the first time. While one ubiquitin is bound in its adenylated form to the active adenylation domain of E1, the second ubiquitin represents the status after transfer and is covalently linked to the active-site cysteine. The covalently linked ubiquitin enables binding of the E2 enzyme without further modification of the ternary Uba1-ubiquitin2 arrangement. This doubly loaded E1 structure constitutes a missing link in the structural analysis of the ubiquitin-transfer cascade.

The radical SAM enzyme AlbA catalyzes thioether bond formation in subtilosin A.

Subtilosin A is a 35-residue, ribosomally synthesized bacteriocin encoded by the sbo-alb operon of Bacillus subtilis. It is composed of a head-to-tail circular peptide backbone that is additionally restrained by three unusual thioether bonds between three cysteines and the α-carbon of one threonine and two phenylalanines, respectively. In this study, we demonstrate that these bonds are synthesized by the radical S-adenosylmethionine enzyme AlbA, which is encoded by the sbo-alb operon and comprises two [4Fe-4S] clusters. One [4Fe-4S] cluster is coordinated by the prototypical CXXXCXXC motif and is responsible for the observed S-adenosylmethionine cleavage reaction, whereas the second [4Fe-4S] cluster is required for the generation of all three thioether linkages. On the basis of the obtained results, we propose a new radical mechanism for thioether bond formation. In addition, we show that AlbA-directed substrate transformation is leader-peptide dependent, suggesting that thioether bond formation is the first step during subtilosin A maturation.

Structural insights into functional modes of proteins involved in ubiquitin family pathways.

The conjugation of ubiquitin and related modifiers to selected proteins represents a general mechanism to alter the function of these protein targets, thereby increasing the complexity of the cellular proteome. Ubiquitylation is catalyzed by a hierarchical enzyme cascade consisting of ubiquitin activating, ubiquitin conjugating, and ubiquitin ligating enzymes, and their combined action results in a diverse topology of ubiquitin-linkages on the modified proteins. Counteracting this machinery are various deubiquitylating enzymes while ubiquitin recognition in all its facets is accomplished by numerous ubiquitin-binding elements. In the following chapter, we attempt to provide an overview on enzymes involved in ubiquitylation as well as the removal of ubiquitin and proteins involved in the recognition and binding of ubiquitin from a structural biologist's perspective.

Dfm1 forms distinct complexes with Cdc48 and the ER ubiquitin ligases and is required for ERAD.

Proteins imported into the endoplasmic reticulum (ER) are scanned for their folding status. Those that do not reach their native conformation are degraded via the ubiquitin-proteasome system. This process is called ER-associated degradation (ERAD). Der1 is known to be one of the components required for efficient degradation of soluble ERAD substrates like CPY(*) (mutated carboxypeptidase yscY). A homologue of Der1 exists, named Dfm1. No function of Dfm1 has been discovered, although a C-terminally hemagglutinin (HA)(3)-tagged Dfm1 protein has been shown to interact with the ERAD machinery. In our studies, we found Dfm1-HA(3) to be an ERAD substrate and therefore not suitable for functional studies of Dfm1 in ERAD. We found cellular, non-tagged Dfm1 to be a stable protein. We identified Dfm1 to be part of complexes which contain the ERAD-L ligase Hrd1/Der3 and Der1 as well as the ERAD-C ligase Doa10. In addition, ERAD of Ste6(*)-HA(3) was strongly dependent on Dfm1. Interestingly, Dfm1 forms a complex with the AAA-ATPase Cdc48 in a strain lacking the Cdc48 membrane-recruiting component Ubx2. This complex does not contain the ubiquitin ligases Hrd1/Der3 and Doa10. The existence of such a complex might point to an additional function of Dfm1 independent from ERAD.

Ubiquitylation in the ERAD Pathway.

Ubiquitylation is a protein modification mechanism, which is found in a multitude of cellular processes like DNA repair and replication, cell signaling, intracellular trafficking and also, very prominently, in selective protein degradation. One specific protein degradation event in the cell concerns the elimination of misfolded proteins to prevent disastrous malfunctioning of cellular pathways. The most complex of these ubiquitylation dependent elimination pathways of misfolded proteins is associated with the endoplasmic reticulum (ER). Proteins, which enter the endoplasmic reticulum for secretion, are folded in this organelle and transported to their site of action. A rigid protein quality control check retains proteins in the endoplasmic reticulum, which fail to fold properly and sends them back to the cytosol for elimination by the proteasome. This requires crossing of the misfolded protein of the endoplasmic reticulum membrane and polyubiquitylation in the cytosol by the ubiquitin-activating, ubiquitin-conjugating and ubiquitin-ligating enzyme machinery.Ubiquitylation is required for different steps of the ER-associated degradation process (ERAD). It facilitates efficient extraction of the ubiquitylated misfolded proteins from and out of the ER membrane by the Cdc48-Ufd1-Npl4 complex and thereby triggers their retro translocation to the cytosol. In addition, the modification with ubiquitin chains guarantees guidance, recognition and binding of the misfolded proteins to the proteasome in the cytosol for efficient degradation.

Sec61p is part of the endoplasmic reticulum-associated degradation machinery.

Endoplasmic reticulum-associated degradation (ERAD) is a cellular pathway for the disposal of misfolded secretory proteins. This process comprises recognition of the misfolded proteins followed by their retro-translocation across the ER membrane into the cytosol in which polyubiquitination and proteasomal degradation occur. A variety of data imply that the protein import channel Sec61p has a function in the ERAD process. Until now, no physical interactions between Sec61p and other essential components of the ERAD pathway could be found. Here, we establish this link by showing that Hrd3p, which is part of the Hrd-Der ubiquitin ligase complex, and other core components of the ERAD machinery physically interact with Sec61p. In addition, we study binding of misfolded CPY(*) proteins to Sec61p during the process of degradation. We show that interaction with Sec61p is maintained until the misfolded proteins are ubiquitinated on the cytosolic side of the ER. Our observations suggest that Sec61p contacts an ERAD ligase complex for further elimination of ER lumenal misfolded proteins.

Ubx4 modulates cdc48 activity and influences degradation of misfolded proteins of the endoplasmic reticulum.

Misfolded proteins of the secretory pathway are recognized in the endoplasmic reticulum (ER), retrotranslocated into the cytoplasm, and degraded by the ubiquitin-proteasome system. Right after retrotranslocation and polyubiquitination, they are extracted from the cytosolic side of the ER membrane through a complex consisting of the AAA ATPase Cdc48 (p97 in mammals), Ufd1, and Npl4. This complex delivers misfolded proteins to the proteasome for final degradation. Extraction, delivery, and processing of ERAD (ER-associated degradation) substrates to the proteasome requires additional cofactors of Cdc48. Here we characterize the UBX domain containing protein Ubx4 (Cui1) as a crucial factor for the degradation of polyubiquitinated proteins via ERAD. Ubx4 modulates the Cdc48-Ufd1-Npl4 complex to guarantee its correct function. Mutant variants of Ubx4 lead to defective degradation of misfolded proteins and accumulation of polyubiquitinated proteins bound to Cdc48. We show the requirement of the UBX domain of Ubx4 for its function in ERAD. The observation that Ubx2 and Ubx4 are not found together in one complex with Cdc48 suggests several distinct steps in modulating the activity and localization of Cdc48 in ERAD.

Ubiquitin ligase Hul5 is required for fragment-specific substrate degradation in endoplasmic reticulum-associated degradation.

To identify new components of the protein quality control and degradation pathway of the endoplasmic reticulum (ER), we performed a growth-based genome-wide screen of about 5000 viable deletion mutants of the yeast Saccharomyces cerevisiae. As substrates we used two misfolded ER membrane proteins, CTL* and Sec61-2L, chimeric derivatives of the classical ER degradation substrates CPY* and Sec61-2. Both substrates contain a cytosolic Leu2 protein fusion, and stabilization of these substrates in ER-associated degradation-deficient strains enables a restored growth of the transformed LEU2-deficient deletion mutants. We identified the strain deleted for the ubiquitin chain elongating ligase Hul5 among the mutant strains with a strong growth phenotype. Here we show that Hul5 is necessary for the degradation of two misfolded ER membrane substrates. Although the degradation of their N-terminal parts is Hul5-independent, the breakdown of their C-terminal fragments requires the ubiquitin chain elongating ligase activity of Hul5. In the absence of Hul5, a truncated form of CTL*myc remains to a large extent embedded in the ER membrane. Hul5 activity promotes the interaction of this truncated CTL*myc with the AAA-ATPase Cdc48, which is known to pull proteins out of the ER membrane. This study unravels the stepwise elimination of the ER membrane-localized CTL*myc substrate. First, N-terminal, lumenal CPY* is transferred to the cytoplasm and degraded by the proteasome. Subsequently, the remaining C-terminal membrane-anchored part requires Hul5 for its effective extraction out of the endoplasmic reticulum and proteasomal degradation.

Aminoacyl-coenzyme A synthesis catalyzed by adenylation domains.

Adenylate forming enzymes play an important role in nature as they are involved in a number of essential biochemical pathways. In this study, we investigated the ability of a set of structurally related recombinant bacterial adenylate forming enzymes derived from nonribosomal peptide synthetases for their ability to synthesize acyl-CoAs in vitro. Adenylation-domains normally transfer their reactive aminoacyl-adenylates onto the covalently attached 4'-phosphopantetheine moiety of small carrier proteins. In detail, DltA, DhbE, GrsA-A, TycB(3)-A, and TycC(3)-A were investigated for their ability to synthesize acyl-CoAs. As reference, acetyl-CoA-synthetase (Acs) of B. subtilis was utilized, which naturally synthesizes acetyl-CoA from acetate, CoA-SH and ATP. Interestingly, all enzymes were capable of producing acyl-CoAs, albeit with differing efficiencies. Surprisingly, both CoA-SH and ATP were observed to inhibit the adenylation reaction at higher concentrations. Product quantification for kinetic determination was carried out by ESI-SIM-MS. Our results allow speculation as to evolutionary relationships within the large class of adenylate forming enzymes.

Yeast genomics in the elucidation of endoplasmic reticulum (ER) quality control and associated protein degradation (ERQD).

The endoplasmic reticulum (ER) is the eukaryotic organelle where most secreted proteins enter the secretory pathway. They enter this organelle in an unfolded state and are folded by a highly active folding machinery to reach their native state. The ER contains an efficient protein quality control system, which recognizes malfolded and orphan proteins and targets them for elimination by a mechanism called ER-associated degradation (ERAD). Both processes are tightly linked, and they will be abbreviated as ERQD (ER quality control and associated degradation). Because ERQD is highly conserved from yeast to man, the easy amenability of yeast to genetic and molecular biological studies combined with the knowledge of its genome and proteome makes it a preferred organism to study such "housekeeping" functions of eukaryotic cells. New genomic and proteomic methods have led to new experimental concepts. Genome-wide screens using genomic deletion libraries led to the identification of genes involved in the processes in question. Using such a genome-wide approach, we devise a sensitive growth test for selection of yeast mutants defective in ERQD. A chimeric protein (CTL*) was generated consisting of the ER luminal, N-glycosylated CPY* protein fused to a transmembrane domain and cytoplasmic 3-isopropylmalate dehydrogenase, the Leu2 protein. In addition, the nonglycosylated ER-membrane-located ERQD substrate Sec61-2p was fused to Leu2p (Sec61-2-L*). Cells carrying a LEU2 deletion can only grow on medium lacking leucine when the chimeric protein CTL* or Sec61-2-L* is not degraded. Thus, only mutant cells defective in an ERQD component can grow. A genome-wide screen can be performed by transforming the CTL* or Sec61-2-L* coding DNA into the approximately 5000 individual deletion mutants of the EUROSCARF yeast library. Examples for new components required for ERQD found by this method are the mannose-6-phosphate receptor domain protein Yos9p and the ubiquitin domain proteins Dsk2p and Rad23p.

Yeast Pex14p possesses two functionally distinct Pex5p and one Pex7p binding sites.

Current evidence favors a cycling receptor model for the import of peroxisomal matrix proteins. The yeast Pex14 protein together with Pex13p and Pex17p form the docking subcomplex at the peroxisomal membrane and interact in this cycle with both soluble import receptors Pex5p and Pex7p. In a first step of a structure-function analysis of Saccharomyces cerevisiae Pex14p, we mapped its binding sites with both receptors. Using the yeast two-hybrid system and pull-down assays, we showed that Pex5p directly interacts with two separate regions of ScPex14p, amino acid residues 1-58 and 235-308. The latter binding site at the C terminus of ScPex14p overlaps with a binding site of Pex7p at amino acid residues 235-325. The functional assessment of these two binding sites of ScPex14p with the peroxisomal targeting signal receptors indicates that they have distinct roles. Deletion of the N-terminal 58 amino acids caused a partial defect of matrix protein import in pex14delta cells expressing the Pex14-(59-341)-p fragment; however, it did not lead to a pex phenotype. In contrast, truncation of the C-terminal 106 amino acids of ScPex14p completely blocked this process. On the basis of these and other published data, we propose that the C terminus of Pex14p contains the actual docking site and discuss the possibility that the N terminus could be involved in a Pex5p-Pex14p association inside the peroxisomal membrane.

Endoplasmic reticulum-associated protein quality control and degradation: genome-wide screen for ERAD components.

In this chapter, a genetic approach is presented that leads to the isolation of mutants and to the identification of proteins involved in protein quality control and endoplasmic reticulum-associated degradation (ERAD). The method makes use of a genomic screen of a yeast deletion library (EUROSCARF). Transformation of each of the approx 5000 strains deleted in one nonvital gene each with a CPY* chimera containing CPY* C-terminally fused to a transmembrane domain and the cytosolic Leu2 protein (3-isopropylmalate dehydrogenase) constitutes the basic screening procedure. Because of a Leu2p deficiency in all deletion strains, cells can grow only when the CTL* chimera is present. As the CPY* module of CTL* will be recognized in ERAD-proficient cells, CTL* will be degraded and the strain is unable to grow. Therefore the absence of genes necessary for ER quality control and ERAD will allow cell growth and indicate the necessity of the respective gene for these processes.

Endoplasmic reticulum-associated protein quality control and degradation: screen for ERAD mutants after ethylmethane sulfonate mutagenesis.

Proteins destined for secretion in eukaryotic cells enter the endoplasmic reticulum (ER) in an unfolded state and are properly folded in this organelle and sent to their final destination. Misfolded or orphan proteins are retained in the ER by a quality control system, retrotranslocated into the cytosol and degraded. Soluble and membrane proteins were found to require a basic machinery for elimination. It is composed of (1) the E1 (ubiquitin activating), E2 (ubiquitin conjugating), and E3 (ubiquitin ligase) enzymes, which polyubiquitinate the substrate proteins during retrotranslocation; (2) the trimeric AAA-ATPase complex Cdc48-Ufd1-Npl4p, which liberates the polyubiquitinated proteins from the ER; and (3) the 26S proteasome, finally degrading the misfolded proteins. Additional components for degradation of soluble or membrane proteins may vary depending on the nature of malfolded proteins. It is therefore of utmost importance to gain insight into the different components of the ER protein quality control and degradation system required for the elimination of the substrate variety. Protein quality control of the ER and subsequent degradation are evolutionarily highly conserved from yeast to human. The yeast Saccharomyces cerevisiae is therefore an elegant model organism for a search of new components of the ER quality control and degradation machinery, because it is easily amenable to genetic and molecular biological experimentation. In this chapter, a genetic approach is presented, which leads to the isolation of mutants and to the identification of proteins involved in protein quality control and ER-associated degradation (ERAD). The method resides in ethylmethane sulfonate (EMS) mutagenesis of a yeast strain followed by screening for stabilization of soluble ERAD substrates, two mutated and consequently malfolded vacuolar enzymes, carboxypeptidase yscY (CPY*) and proteinase yscA (PrA*). Both malfolded proteins are retained in the ER lumen and become substrates of the ERAD machinery.

Functional similarity between the peroxisomal PTS2 receptor binding protein Pex18p and the N-terminal half of the PTS1 receptor Pex5p.

Within the extended receptor cycle of peroxisomal matrix import, the function of the import receptor Pex5p comprises cargo recognition and transport. While the C-terminal half (Pex5p-C) is responsible for PTS1 binding, the contribution of the N-terminal half of Pex5p (Pex5p-N) to the receptor cycle has been less clear. Here we demonstrate, using different techniques, that in Saccharomyces cerevisiae Pex5p-N alone facilitates the import of the major matrix protein Fox1p. This finding suggests that Pex5p-N is sufficient for receptor docking and cargo transport into peroxisomes. Moreover, we found that Pex5p-N can be functionally replaced by Pex18p, one of two auxiliary proteins of the PTS2 import pathway. A chimeric protein consisting of Pex18p (without its Pex7p binding site) fused to Pex5p-C is able to partially restore PTS1 protein import in a PEX5 deletion strain. On the basis of these results, we propose that the auxiliary proteins of the PTS2 import pathway fulfill roles similar to those of the N-terminal half of Pex5p in the PTS1 import pathway.

Pex15p of Saccharomyces cerevisiae provides a molecular basis for recruitment of the AAA peroxin Pex6p to peroxisomal membranes.

The gene products (peroxins) of at least 29 PEX genes are known to be necessary for peroxisome biogenesis but for most of them their precise function remains to be established. Here we show that Pex15p, an integral peroxisomal membrane protein, in vivo and in vitro binds the AAA peroxin Pex6p. This interaction functionally interconnects these two hitherto unrelated peroxins. Pex15p provides the mechanistic basis for the reversible targeting of Pex6p to peroxisomal membranes. We could demonstrate that the N-terminal part of Pex6p contains the binding site for Pex15p and that the two AAA cassettes D1 and D2 of Pex6p have opposite effects on this interaction. A point mutation in the Walker A motif of D1 (K489A) decreased the binding of Pex6p to Pex15p indicating that the interaction of Pex6p with Pex15p required binding of ATP. Mutations in Walker A (K778A) and B (D831Q) motifs of D2 abolished growth on oleate and led to a considerable larger fraction of peroxisome bound Pex6p. The nature of these mutations suggested that ATP-hydrolysis is required to disconnect Pex6p from Pex15p. On the basis of these results, we propose that Pex6p exerts at least part of its function by an ATP-dependent cycle of recruitment and release to and from Pex15p.

Characterization of a new type of phosphopantetheinyl transferase for fatty acid and siderophore synthesis in Pseudomonas aeruginosa.

Phosphopantetheinyl-dependent carrier proteins are part of fatty-acid synthases (primary metabolism), polyketide synthases, and non-ribosomal peptide synthetases (secondary metabolism). For these proteins to become functionally active, they need to be primed with the 4'-phosphopantetheine moiety of coenzyme A by a dedicated phosphopantetheine transferase (PPTase). Most organisms that employ more than one phosphopantetheinyl-dependent pathway also have more than one PPTase. Typically, one of these PPTases is optimized for the modification of carrier proteins of primary metabolism and rejects those of secondary metabolism (AcpS-type PPTases), whereas the other, Sfp-type PPTase, efficiently modifies carrier proteins involved in secondary metabolism. We present here a new type of PPTase, the carrier protein synthase of Pseudomonas aeruginosa, an organism that harbors merely one PPTase, namely PcpS. Gene deletion experiments clearly show that PcpS is essential for growth of P. aeruginosa, and biochemical data indicate its association with both fatty acid synthesis and siderophore metabolism. At first sight, PcpS is a PPTase of the monomeric Sfp-type and was consequently expected to have catalytic properties typical for this type of enzyme. However, in vitro characterization of PcpS with natural protein partners and non-cognate substrates revealed that its catalytic properties differ significantly from those of Sfp. Thus, the situation in P. aeruginosa is not simply the result of the loss of an AcpS-type PPTase. PcpS exhibits high catalytic efficiency with the carrier protein of fatty acid synthesis and shows a reduced although significant conversion rate of the carrier proteins of non-ribosomal peptide synthetases from their apo to holo form. This association with enzymes of primary and secondary metabolism indicates that PcpS belongs to a new sub-class of PPTases.