Tsc10p and FVT1: topologically distinct short-chain reductases required for long-chain base synthesis in yeast and mammals

In yeast, Tsc10p catalyzes reduction of 3-ketosphinganine to dihydrosphingosine. In mammals, it has been proposed that this reaction is catalyzed by FVT1, which despite limited homology and a different predicted topology, can replace Tsc10p in yeast. Silencing of FVT1 revealed a direct correlation between FVT1 levels and reductase activity, showing that FVT1 is the principal 3-ketosphinganine reductase in mammalian cells. Localization and topology studies identified an N-terminal membrane-spanning domain in FVT1 (absent in Tsc10p) oriented to place it in the endoplasmic reticulum (ER) lumen. In contrast, protease digestion studies showed that the N terminus of Tsc10p is cytoplasmic. Fusion of the N-terminal domain of FVT1 to green fluorescent protein directed the fusion protein to the ER, demonstrating that it is sufficient for targeting. Although both proteins have two predicted transmembrane domains C-terminal to a cytoplasmic catalytic domain, neither had an identifiable lumenal loop. Nevertheless, both Tsc10p and the residual fragment of FVT1 produced by removal of the N-terminal domain with factor Xa protease behave as integral membrane proteins. In addition to their topological differences, mutation of conserved catalytic residues had different effects on the activities of the two enzymes. Thus, while FVT1 can replace Tsc10p in yeast, there are substantial differences between the two enzymes that may be important for regulation of sphingolipid biosynthesis in higher eukaryotes.     

The Drosophila RBP-J kappa gene encodes the binding protein for the immunoglobulin J kappa recombination signal sequence.

We previously isolated a cDNA encoding the 60-kDa murine protein (RBP-J kappa protein) that specifically binds to the immunoglobulin J kappa recombination signal sequence. The RBP-J kappa gene is highly conserved in a wide variety of organisms including man, Xenopus, Drosophila, and yeast. We have isolated and characterized the Drosophila homologue of the RBP-J kappa gene. The Drosophila RBP-J kappa gene was mapped to the polytene region 35BC of chromosome 2. The nucleotide sequence of this gene indicates that it is not one of the known genes located in the 35 BC region. The nucleotide and amino acid sequences of the Drosophila and mouse RBP-J kappa genes are 60 and 75% homologous, respectively. The central 248-residue regions of RBP-J kappa proteins of the two species are 93% homologous and include the 40-residue integrase motif. The Drosophila RBP-J kappa protein expressed in COS cells bound to the J kappa recognition sequence with the same specificity as the murine counterpart. These results suggest that Drosophila may have a site-specific recombination system which utilizes the immunoglobulin recombination signal sequence. Implications for evolution of immunoglobulin gene rearrangement were also discussed.     

The yeast ubiquitin genes: a family of natural gene fusions.

Ubiquitin is a 76-residue protein highly conserved among eukaryotes. Conjugation of ubiquitin to intracellular proteins mediates their selective degradation in vivo. We describe a family of four ubiquitin-coding loci in the yeast Saccharomyces cerevisiae. UB11, UB12 and UB13 encode hybrid proteins in which ubiquitin is fused to unrelated ('tail') amino acid sequences. The ubiquitin coding elements of UB11 and UB12 are interrupted at identical positions by non-homologous introns. UB11 and UB12 encode identical 52-residue tails, whereas UB13 encodes a different 76-residue tail. The tail amino acid sequences are highly conserved between yeast and mammals. Each tail contains a putative metal-binding, nucleic acid-binding domain of the form Cys-X2-4-Cys-X2-15-Cys-X2-4-Cys, suggesting that these proteins may function by binding to DNA. The fourth gene, UB14, encodes a polyubiquitin precursor protein containing five ubiquitin repeats in a head-to-tail, spacerless arrangement. All four ubiquitin genes are expressed in exponentially growing cells, while in stationary-phase cells the expression of UB11 and UB12 is repressed. The UB14 gene, which is strongly inducible by starvation, high temperatures and other stresses, contains in its upstream region strong homologies to the consensus 'heat shock box' nucleotide sequence. Elsewhere we show that the essential function of the UB14 gene is to provide ubiquitin to cells under stress.     

Structural Conservation of Components in the Amino Acid Sensing Branch of the TOR Pathway in Yeast and Mammals

The highly conserved Rag family GTPases have a role in reporting amino acid availability to the TOR (target of rapamycin) signaling complex, which regulates cell growth and metabolism in response to environmental cues. The yeast Rag proteins Gtr1p and Gtr2p were shown in multiple independent studies to interact with the membrane-associated proteins Gse1p (Ego3p) and Gse2p (Ego1p). However, mammalian orthologs of Gse1p and Gse2p could not be identified. We determined the crystal structure of Gse1p and found it to match the fold of two mammalian proteins, MP1 (mitogen-activated protein kinase scaffold protein 1) and p14, which form a heterodimeric complex that had been assigned a scaffolding function in mitogen-activated protein kinase pathways. The significance of this structural similarity is validated by the recent identification of a physical and functional association between mammalian Rag proteins and MP1/p14. Together, these findings reveal that key components of the TOR signaling pathway are structurally conserved between yeast and mammals, despite divergence of sequence to a degree that thwarts detection through simple homology searches.     

Identification of the proteins required for biosynthesis of diphthamide, the target of bacterial ADP-ribosylating toxins on translation elongation factor 2

Diphthamide, a posttranslational modification of translation elongation factor 2 that is conserved in all eukaryotes and archaebacteria and is the target of diphtheria toxin, is formed in yeast by the actions of five proteins, Dph1 to -5, and a still unidentified amidating enzyme. Dph2 and Dph5 were previously identified. Here, we report the identification of the remaining three yeast proteins (Dph1, -3, and -4) and show that all five Dph proteins have either functional (Dph1, -2, -3, and -5) or sequence (Dph4) homologs in mammals. We propose a unified nomenclature for these proteins (e.g., HsDph1 to -5 for the human proteins) and their genes based on the yeast nomenclature. We show that Dph1 and Dph2 are homologous in sequence but functionally independent. The human tumor suppressor gene OVCA1, previously identified as homologous to yeast DPH2, is shown to actually be HsDPH1. We show that HsDPH3 is the previously described human diphtheria toxin and Pseudomonas exotoxin A sensitivity required gene 1 and that DPH4 encodes a CSL zinc finger-containing DnaJ-like protein. Other features of these genes are also discussed. The physiological function of diphthamide and the basis of its ubiquity remain a mystery, but evidence is presented that Dph1 to -3 function in vivo as a protein complex in multiple cellular processes.     

Cloning and comparative sequence analysis of PUM1 and PUM2 genes,human members of the Pumilio family of RNA-binding proteins

Drosophila gene Pumilio (Pum) is a founder member of an evolutionarily conserved family of RNA-binding proteins that are present from yeast to mammals, and act as translational repressors during embryo development and cell differentiation. The human genome contains two Pumilio related genes, PUM1 and PUM2, that encode 127 and 114 kDa proteins with evolutionarily highly conserved Pum RNA-binding domain (86 and 88% homology with the fly Pum protein). PUM1 and PUM2 proteins share 83% overall similarity, with RNA-binding domain being 91% identical. Both PUM1 and PUM2 show relatively widespread and mostly overlapping expression in human tissues, and are very large genes with highly conserved gene structure. PUM1 consists of 22 exons, spanning about 150 kb on chromosome 1p35.2, whereas PUM2 consists of 20 exons and spans at least 80 kb on chromosome 2p23-24. Extremely high evolutionary conservation of the RNA-binding domain from yeast to humans, and conserved function of Pumilio proteins in invertebrates and lower vertebrates suggest that mammalian Pumilio proteins could also play an important role in translational regulation of embryogenesis and cell development and differentiation.     

The Rheb family of GTP-binding proteins

Rheb proteins represent a novel and unique family of the Ras superfamily GTP-binding proteins that is conserved from yeast to human. Biochemical studies establish that they bind and hydrolyze GTP. Molecular modeling studies reveal a few structural differences between Rheb and Ras, which may suggest that residues involved in biochemical activities differ between the two G-proteins. The function of Rheb has been studied in a number of organisms that point to the involvement of Rheb in cell growth and cell cycle progression. In addition, studies in fungi suggest that Rheb is involved in arginine uptake. Further studies in Drosophila and mammalian cells have shown that the effects of Rheb on growth and cell cycle progression are mediated by the effect on the insulin/TOR/S6K signaling pathway. These studies have also shown that a complex consisting of the tuberous sclerosis gene products, Tsc1/Tsc2, serves as a GTPase activating protein (GAP) for Rheb, implying Rheb's role in this genetic disorder. Finally, Rheb proteins have been shown to be farnesylated and small molecule inhibitors of protein farnesyltransferase can block the ability of Rheb to activate the TOR/S6K signaling.     

The human SWI-SNF complex protein p270 is an ARID family member with non-sequence-specific DNA binding activity

p270 is an integral member of human SWI-SNF complexes, first identified through its shared antigenic specificity with p300 and CREB binding protein. The deduced amino acid sequence of p270 reported here indicates that it is a member of an evolutionarily conserved family of proteins distinguished by the presence of a DNA binding motif termed ARID (AT-rich interactive domain). The ARID consensus and other structural features are common to both p270 and yeast SWI1, suggesting that p270 is a human counterpart of SWI1. The approximately 100-residue ARID sequence is present in a series of proteins strongly implicated in the regulation of cell growth, development, and tissue-specific gene expression. Although about a dozen ARID proteins can be identified from database searches, to date, only Bright (a regulator of B-cell-specific gene expression), dead ringer (a Drosophila melanogaster gene product required for normal development), and MRF-2 (which represses expression from the cytomegalovirus enhancer) have been analyzed directly in regard to their DNA binding properties. Each binds preferentially to AT-rich sites. In contrast, p270 shows no sequence preference in its DNA binding activity, thereby demonstrating that AT-rich binding is not an intrinsic property of ARID domains and that ARID family proteins may be involved in a wider range of DNA interactions.     

p62/SQSTM1 binds directly to Atg8/LC3 to facilitate degradation of ubiquitinated protein aggregates by autophagy

Protein degradation by basal constitutive autophagy is important to avoid accumulation of polyubiquitinated protein aggregates and development of neurodegenerative diseases. The polyubiquitin-binding protein p62/SQSTM1 is degraded by autophagy. It is found in cellular inclusion bodies together with polyubiquitinated proteins and in cytosolic protein aggregates that accumulate in various chronic, toxic, and degenerative diseases. Here we show for the first time a direct interaction between p62 and the autophagic effector proteins LC3A and -B and the related gamma-aminobutyrate receptor-associated protein and gamma-aminobutyrate receptor-associated-like proteins. The binding is mediated by a 22-residue sequence of p62 containing an evolutionarily conserved motif. To monitor the autophagic sequestration of p62- and LC3-positive bodies, we developed a novel pH-sensitive fluorescent tag consisting of a tandem fusion of the red, acid-insensitive mCherry and the acid-sensitive green fluorescent proteins. This approach revealed that p62- and LC3-positive bodies are degraded in autolysosomes. Strikingly, even rather large p62-positive inclusion bodies (2 microm diameter) become degraded by autophagy. The specific interaction between p62 and LC3, requiring the motif we have mapped, is instrumental in mediating autophagic degradation of the p62-positive bodies. We also demonstrate that the previously reported aggresome-like induced structures containing ubiquitinated proteins in cytosolic bodies are dependent on p62 for their formation. In fact, p62 bodies and these structures are indistinguishable. Taken together, our results clearly suggest that p62 is required both for the formation and the degradation of polyubiquitin-containing bodies by autophagy.     

The integrase family of site-specific recombinases: regional similarities and global diversity.

A combination of two methods for detecting distant relationships in protein primary sequences was used to compare the site-specific recombination proteins encoded by bacteriophage lambda, phi 80, P22, P2, 186, P4 and P1. This group of proteins exhibits an unexpectedly large diversity of sequences. Despite this diversity, all of the recombinases can be aligned in their C-terminal halves. A 40-residue region near the C terminus is particularly well conserved in all the proteins and is homologous to a region near the C terminus of the yeast 2 mu plasmid Flp protein. This family of recombinases does not appear to be related to any other site-specific recombinases. Three positions are perfectly conserved within this family: histidine, arginine and tyrosine are found at respective alignment positions 396, 399 and 433 within the well-conserved C-terminal region. We speculate that these residues contribute to the active site of this family of recombinases, and suggest that tyrosine-433 forms a transient covalent linkage to DNA during strand cleavage and rejoining.     

Biochemical studies of Zmpste24-deficient mice

Genetic studies in Saccharomyces cerevisiae identified two genes, STE24 and RCE1, involved in cleaving the three carboxyl-terminal amino acids from isoprenylated proteins that terminate with a CAAX sequence motif. Ste24p cleaves the carboxyl-terminal "-AAX" from the yeast mating pheromone a-factor, whereas Rce1p cleaves the -AAX from both a-factor and Ras2p. Ste24p also cleaves the amino terminus of a-factor. The mouse genome contains orthologues for both yeast RCE1 and STE24. We previously demonstrated, with a gene-knockout experiment, that mouse Rce1 is essential for development and that Rce1 is entirely responsible for the carboxyl-terminal proteolytic processing of the mouse Ras proteins. In this study, we cloned mouse Zmpste24, the orthologue for yeast STE24 and showed that it could promote a-factor production when expressed in yeast. Then, to assess the importance of Zmpste24 in development, we generated Zmpste24-deficient mice. Unlike the Rce1 knockout mice, Zmpste24-deficient mice survived development and were fertile. Since no natural substrates for mammalian Zmpste24 have been identified, yeast a-factor was used as a surrogate substrate to investigate the biochemical activities in membranes from the cells and tissues of Zmpste24-deficient mice. We demonstrate that Zmpste24-deficient mouse membranes, like Ste24p-deficient yeast membranes, have diminished CAAX proteolytic activity and lack the ability to cleave the amino terminus of the a-factor precursor. Thus, both enzymatic activities of yeast Ste24p are conserved in mouse Zmpste24, but these enzymatic activities are not essential for mouse development or for fertility.     

Two‐state conformational equilibrium in the Par‐4 leucine zipper domain

Prostate apoptosis response factor‐4 (Par‐4) is a pro‐apoptotic and tumor‐suppressive protein. A highly conserved heptad repeat sequence at the Par‐4 C‐terminus suggests the presence of a leucine zipper (LZ). This C‐terminal region is essential for Par‐4 self‐association and interaction with various effector proteins. We have used nuclear magnetic resonance (NMR) spectroscopy to fully assign the chemical shift resonances of a peptide comprising the LZ domain of Par‐4 at neutral pH. Further, we have investigated the properties of the Par‐4 LZ domain and two point mutants under a variety of conditions using NMR, circular dichroism (CD), light scattering, and bioinformatics. Results indicate an environment‐dependent conformational equilibrium between a partially ordered monomer (POM) and a predominantly coiled coil dimer (CCD). The combination of techniques used allows the time scales of the equilibrium to be probed and also helps to identify features of the amino acid sequence that may influence the equilibrium. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

Structures of yeast vesicle trafficking proteins

In protein transport between organelles, interactions of v- and t-SNARE proteins are required for fusion of protein-containing vesicles with appropriate target compartments. Mammalian SNARE proteins have been observed to interact with NSF and SNAP, and yeast SNAREs with yeast homologues of NSF and SNAP proteins. This observation led to the hypothesis that, despite low sequence homology, SNARE proteins are structurally similar among eukaryotes. SNARE proteins can be classified into two groups depending on whether they interact with SNARE binding partners via conserved glutamine (Q-SNAREs) or arginine (R-SNAREs). Much of the published structural data available is for SNAREs involved in exocytosis (either in yeast or synaptic vesicles). This paper describes circular dichroism, Fourier transform infrared spectroscopy, and dynamic light scattering data for a set of yeast v- and t-SNARE proteins, Vti1p and Pep12p, that are Q-SNAREs involved in intracellular trafficking. Our results suggest that the secondary structure of Vti1p is highly alpha-helical and that Vti1p forms multimers under a variety of solution conditions. In these respects, Vti1p appears to be distinct from R-SNARE proteins characterized previously. The alpha-helicity of Vti1p is similar to that of Q-SNARE proteins characterized previously. Pep12p, a Q-SNARE, is highly alpha-helical. It is distinct from other Q-SNAREs in that it forms dimers under many of the solution conditions tested in our experiments. The results presented in this paper are among the first to suggest heterogeneity in the functioning of SNARE complexes.     

Targeting of Tsc13p to nucleus-vacuole junctions: a role for very-long-chain fatty acids in the biogenesis of microautophagic vesicles

TSC13 is required for the biosynthesis of very-long-chain fatty acids (VLCFAs) in yeast. Tsc13p is a polytopic endoplasmic reticulum (ER) membrane protein that accumulates at nucleus-vacuole (NV) junctions, which are formed through Velcro-like interactions between Nvj1p in the perinuclear ER and Vac8p on the vacuole membrane. NV junctions mediate piecemeal microautophagy of the nucleus (PMN), during which bleb-like portions of the nucleus are extruded into invaginations of the vacuole membrane and degraded in the vacuole lumen. We report that Tsc13p is sequestered into NV junctions from the peripheral ER through Vac8p-independent interactions with Nvj1p. During nutrient limitation, Tsc13p is incorporated into PMN vesicles in an Nvj1p-dependent manner. The lumenal diameters of PMN blebs and vesicles are significantly reduced in tsc13-1 and tsc13-1 elo3-Delta mutant cells. PMN structures are also smaller in cells treated with cerulenin, an inhibitor of de novo fatty acid synthesis and elongation. The targeting of Tsc13p-GFP into NV junctions is perturbed by cerulenin, suggesting that its binding to Nvj1p depends on the availability of fatty acid substrates. These results indicate that Nvj1p retains and compartmentalizes Tsc13p at NV junctions and that VLCFAs contribute to the normal biogenesis of trilaminar PMN structures in yeast.     

The role of the LH subdomain in the function of the Cip/Kip cyclin-dependent kinase regulators

The Cip/Kip protein family, which includes p27, p21, and p57, modulates the activity of cyclin-dependent kinases (Cdks). A domain within these proteins, termed the kinase inhibitory domain (KID), is necessary and sufficient for Cdk inhibition. The KID consists of a cyclin-binding subdomain (termed D1) and a Cdk-binding subdomain (termed D2) joined by a 22-residue linker subdomain (termed LH). Before binding the Cdks, D1 and D2 are largely unstructured and the LH subdomain exhibits nascent helical characteristics. Curiously, although the sequence of the linker subdomain is not highly conserved within the family, its nascent helical structure is conserved. In this study, we explored the role of this structural conservation in interactions with cyclin-dependent kinase 2 (Cdk2) and cyclin A. We constructed chimeric p27-KID molecules in which the p27 LH subdomain was replaced with the corresponding segments of either p21 or p57. The chimeric molecules bind and inhibit Cdk2 in a manner similar to wild-type p27-KID. However, the extent of enthalpy/entropy compensation associated with these interactions was dramatically different, indicating different extents of LH subdomain folding upon binding. Our results indicate that the different LH subdomains, despite their sequence and thermodynamic differences, play similar roles in binding and inhibiting Cdk2/cyclin A.     

Exploration of yeast alkali metal cation/H+ antiporters: Sequence and structure comparison

The Saccharomyces cerevisiae genome contains three genes encoding alkali metal cation/H+ antiporters (Nha1p, Nhx1p, Kha1p) that differ in cell localization, substrate specificity and physiological function. Systematic genome sequencing of other yeast species revealed highly conserved homologous ORFs in all of them. We compared the yeast sequences both at DNA and protein levels. The subfamily of yeast endosomal/prevacuolar Nhx1 antiporters is closely related to mammalian plasma membrane NHE proteins and to both plasma membrane and vacuolar plant antiporters. The high sequence conservation within this subfamily of yeast antiporters suggests that Nhx1p is of great importance in cell physiology. Yeast Kha1 proteins probably belong to the same subfamily as bacterial antiporters, whereas Nhal proteins form a distinct subfamily.     

Mutational analysis of conserved sequence motifs in the budding yeast Cdc6 protein

The Cdc6 protein is required to load a complex of Mcm2-7 family members (the MCM complex) into prereplicative complexes at budding yeast origins of DNA replication. Cdc6p is a member of the AAA(+) superfamily of proteins, which includes the prokaryotic and eukaryotic clamp loading proteins. These proteins share a number of conserved regions of homology and a common three-dimensional architecture. Two of the conserved sequence motifs are the Walker A and B motifs that are involved in nucleotide metabolism and are essential for Cdc6p function in vivo. Here, we analyse mutants in the other conserved sequence motifs. Several of these mutants are temperature-sensitive for growth and are unable to recruit the MCM complex to chromatin at the restrictive temperature. In one such temperature-sensitive mutant, a highly conserved asparagine residue in the sensor I motif was changed to alanine. Overexpression of this mutant protein is lethal. This phenotype is very similar to the phenotype previously described for a mutation in the Walker B motif, suggesting a common role for sensor I and the Walker B motif in Cdc6 function.     

Characterization of pancreatic ERj3p, a homolog of yeast DnaJ-like protein Scj1p

We have previously identified in the human EST sequence data base four overlapping clones that could be aligned with both a predicted protein sequence, deduced from the C. elegans genomic sequence, and partial amino acid sequences, obtained for a protein from canine pancreatic microsomes. We suggested that these proteins are homologs of yeast microsomal and DnaJ-like protein Scj1p and termed them ERj3p. Here we verified the predicted protein sequence of human ERj3p by sequence analysis of the corresponding cDNA. Multiple alignment of related sequences identified these proteins as true homologs of yeast Scj1p. Biochemical analysis of the canine protein characterized ERj3p as a soluble glycoprotein of the pancreatic endoplasmic reticulum. This pancreatic DnaJ-like protein was shown to interact with lumenal DnaK-like proteins, such as BiP. Furthermore, we found that ERj3p interacts with SDF2L1 protein that may be involved in protein O-glycosylation. We propose that ERj3p represents a cochaperone of DnaK-like chaperones of the mammalian endoplasmic reticulum and is involved in folding and maturation of newly synthesized proteins.