206 research outputs found
Cytoplasmic N-Terminal Protein Acetylation Is Required for Efficient Photosynthesis in Arabidopsis
No full textThe Arabidopsis atmak3-1 mutant was identified on the basis of a decreased effective quantum yield of photosystem II. In atmak3-1, the synthesis of the plastome-encoded photosystem II core proteins D1 and CP47 is affected, resulting in a decrease in the abundance of thylakoid multiprotein complexes. DNA array-based mRNA analysis indicated that extraplastid functions also are altered. The mutation responsible was localized to AtMAK3, which encodes a homolog of the yeast protein Mak3p. In yeast, Mak3p, together with Mak10p and Mak31p, forms the N-terminal acetyltransferase complex C (NatC). The cytoplasmic AtMAK3 protein can functionally replace Mak3p, Mak10p, and Mak31p in acetylating N termini of endogenous proteins and the L-A virus Gag protein. This result, together with the finding that knockout of the Arabidopsis MAK10 homolog does not result in obvious physiological effects, indicates that AtMAK3 function does not require NatC complex formation, as it does in yeast. We suggest that N-acetylation of certain chloroplast precursor protein(s) is necessary for the efficient accumulation of the mature protein(s) in chloroplasts
Expression of a yeast superkiller gene(SKI3) in Saccharomyces cerevisiae
No full textA yeast chromosomal superkiller gene (SKI3) was cloned and expressed in ski-Saccharomyces cerevisiae strains. The gene was fused to the structural region of E. coli lacZ gene at its C-terminus in a yeast-E. coli shuttle vector, pSR605. The fused gene complemented ski3-strains with SKI3 activity and the quantitative level of expression was measured as determined by assaying Beta-galactosidase activity. The SDS-polyacrylamide gel electrophoresis and the Western blot analysis of this fused protein showed the immuno-reacted bands with a protein of the estimated molecular size (ca.250 Kd).open
A region of proto-dbl essential for its transforming activity shows sequence similarity to a yeast cell cycle gene, CDC24, and the human breakpoint cluster gene, bcr
No full textProto-dbl is a human proto-oncogene, whose oncogenic activation was initially detected by DNA transfection. We report significant sequence similarity between the predicted proto-dbl product and the products of CDC24, a Saccharomyces cerevisiae cell division cycle gene required for correct budding and establishment of cell polarity, and bcr, a gene implicated in the pathogenesis of chronic myelogenous leukemia (CML). Of 925 residues of the predicted proto-dbl protein, a stretch of 238 residues showed 29% and 22% identity over a region of similar length of the CDC24 and bcr proteins, respectively. When evolutionarily conservative substitutions were taken into account, the similarities were 68.8% and 71.6% for proto-dbl/CDC24 and proto-dbl/bcr gene products, respectively. Moreover, all three sequences were predicted to be markedly hydrophilic over this region. Very small deletions within the conserved region completely abolished transforming activity of dbl, while extensive deletion outside of this region had no effect. Even substitutions over a small stretch of close similarity with the other proteins substantially impaired transforming activity. Cells transformed by the dbl oncogene, like cdc24 mutants arrested at the nonpermissive temperature, form multinucleate cells. Thus, our findings indicate that the conserved region is an essential domain that may reflect important functional similarities among these otherwise highly divergent molecules
Prion Seeds Distribute throughout the Eyes of Sporadic Creutzfeldt-Jakob Disease Patients
No full textSporadic Creutzfeldt-Jakob disease (sCJD) is the most common prion disease in humans and has been iatrogenically transmitted through corneal graft transplantation. Approximately 40% of sCJD patients develop visual or oculomotor symptoms and may seek ophthalmological consultation. Here we used the highly sensitive real-time quaking-induced conversion (RT-QuIC) assay to measure postmortem prion seeding activities in cornea, lens, ocular fluid, retina, choroid, sclera, optic nerve, and extraocular muscle in the largest series of sCJD patient eyes studied by any assay to date. We detected prion seeding activity in 100% of sCJD eyes, representing three common sCJD subtypes, with levels varying by up to 4 log-fold among individuals. The retina consistently showed the highest seed levels, which in some cases were only slightly lower than brain. Within the retina, prion deposits were detected by immunohistochemistry (IHC) in the retinal outer plexiform layer in most sCJD cases, and in some eyes the inner plexiform layer, consistent with synaptic prion deposition. Prions were not detected by IHC in any other eye region. With RT-QuIC, prion seed levels generally declined in eye tissues with increased distance from the brain, and yet all corneas had prion seeds detectable. Prion seeds were also present in the optic nerve, extraocular muscle, choroid, lens, vitreous, and sclera. Collectively, these results reveal that sCJD patients accumulate prion seeds throughout the eye, indicating the potential diagnostic utility as well as a possible biohazard.IMPORTANCE Cases of iatrogenic prion disease have been reported from corneal transplants, yet the distribution and levels of prions throughout the eye remain unknown. This study probes the occurrence, level, and distribution of prions in the eyes of patients with sporadic Creutzfeldt-Jakob disease (sCJD). We tested the largest series of prion-infected eyes reported to date using an ultrasensitive technique to establish the prion seed levels in eight regions of the eye. All 11 cases had detectable prion seeds in the eye, and in some cases, the seed levels in the retina approached those in brain. In most cases, prion deposits could also be seen by immunohistochemical staining of retinal tissue; other ocular tissues were negative. Our results have implications for estimating the risk for iatrogenic transmission of sCJD as well as for the development of antemortem diagnostic tests for prion diseases
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No full textLike other intracellular fusion events, the homotypic fusion of yeast vacuoles requires a Rab GTPase, a large Rab effector complex, SNARE proteins which can form a 4-helical bundle, and the SNARE disassembly chaperones Sec17p and Sec18p. In addition to these proteins, specific vacuole lipids are required for efficient fusion in vivo and with the purified organelle. Reconstitution of vacuole fusion with all purified components reveals that high SNARE levels can mask the requirement for a complex mixture of vacuole lipids. At lower, more physiological SNARE levels, neutral lipids with small headgroups that tend to form non-bilayer structures (phosphatidylethanolamine, diacylglycerol, and ergosterol) are essential. Membranes without these three lipids can dock and complete trans -SNARE pairing but cannot rearrange their lipids for fusion
A short region upstream of the yeast vacuolar Qa-SNARE heptad-repeats promotes membrane fusion through enhanced SNARE complex assembly
No full textWhereas SNARE (soluble N-ethylmaleimide–sensitive factor attachment protein receptor) heptad-repeats are well studied, SNAREs also have upstream N-domains of indeterminate function. The assembly of yeast vacuolar SNAREs into complexes for fusion can be studied in chemically defined reactions. Complementary proteoliposomes bearing a Rab:GTP and either the vacuolar R-SNARE or one of the three integrally anchored Q-SNAREs were incubated with the tethering/SM protein complex HOPS and the two other soluble SNAREs (lacking a transmembrane anchor) or their SNARE heptad-repeat domains. Fusion required a transmembrane-anchored R-SNARE on one membrane and an anchored Q-SNARE on the other. The N-domain of the Qb-SNARE was completely dispensable for fusion. Whereas fusion can be promoted by very high concentrations of the Qa-SNARE heptad-repeat domain alone, at physiological concentrations the Qa-SNARE heptad-repeat domain alone has almost no fusion activity. The 181–198 region of Qa, immediately upstream of the SNARE heptad-repeat domain, is required for normal fusion activity with HOPS. This region is needed for normal SNARE complex assembly.</jats:p
N-Terminal Domain of Vacuolar SNARE Vam7p Promotes Trans-SNARE Complex Assembly
No full textSNARE-dependent membrane fusion in eukaryotic cells requires that the heptad-repeat SNARE domains from R- and Q-SNAREs, anchored to apposed membranes, assemble into four-helix coiled-coil bundles. In addition to their SNARE and transmembrane domains, most SNAREs have N-terminal domains (N-domains), although their functions are unclear. The N-domain of the yeast vacuolar Qc-SNARE Vam7p is a binding partner for the homotypic fusion and vacuole protein sorting complex (a master regulator of vacuole fusion) and has Phox homology, providing a phosphatidylinositol 3-phosphate (PI3P)-specific membrane anchor. We now report that this Vam7p N-domain has yet another role, one that does not depend on its physical connection to the Vam7p SNARE domain. By attaching a transmembrane anchor to the C terminus of Vam7p to create Vam7tm, we bypass the requirement for the N-domain to anchor Vam7tm to reconstituted proteoliposomes. The N-domain of Vam7tm is indispensible for trans-SNARE complex assembly in SNARE-only reactions. Introducing Vam7(1-125)p as a separate recombinant protein suppresses the defect caused by N-domain deletion from Vam7tm, demonstrating that the function of this N-domain is not constrained to covalent attachment to Vam7p. The Vam7p N-domain catalyzes the docking of apposed membranes by promoting transinteractions between R- and Q-SNAREs. This function of the Vam7p N-domain depends on the presence of PI3P and its affinity for PI3P. Added N-domain can even promote SNARE complex assembly when Vam7 still bears its own N-domain
The Family of Small Heat Shock Proteins: Assembly and Binding Functions
No full textSmall heat shock proteins (sHsps) are a diverse family evolved from ancient stress proteins that are now associated with protecting microbial, plant, worm, insect and mammalian cells under stress, where prevention of aggregation of destabilized proteins is considered to be their general function. The sequences all contain a short a-crystallin domain (ACD) bracketed by variable sequence extensions that contribute to a dynamic continuum of oligomeric forms. A few large symmetric oligomeric assemblies from nonmetazoans have been resolved by crystallography, as well as a few metazoan subassembly forms. A structure-based sequence alignment of the ACDs from resolved structures with representative sequences of sHsps from species of high economic, model or medical value is presented. The ACDs are portrayed to emphasize their interaction interfaces. The construction of a range of nanoassemblies from a polyvalent building block by binding IXI/V sequence motifs in extensions into partner ACD pockets is illustrated. This interaction mechanism can also be used to connect sHsps to energy driven chaperone machines, as shown for the interaction with cochaperone Bag3 in myofibril Z-disks. The resolved complete assemblies are built from a strand exchange dimer, whereas animal sHsps have an antiparallel (AP) dimer interface that forms a shared groove which can change shape. Spectroscopy has shown how the AP interface is regulated by pH. Animal sHsp full assemblies are undetermined, although some clues as to the structural role of the hydrophobic N-terminal extensions can be surmised from analysis of the resolved assemblies. A mechanism for prevention of protein aggregation is likely to involve a stress-regulated ensemble of interconverting destabilized sHsp assemblies when interfaces and extensions become more accessible to the proteome. Rapid developments in EM offer the possibility of resolution of an sHsp ensemble if the level of polydispersity can be lowered
Protein-DNA interactions at the origin of simian virus 40 DNA replication
No full textThe processes involved in initiating DNA replication have recently been studied with great success. The major advances have largely come from in vitro studies of simple bacteriophage systems such as those described in this volume (Meyer et al.; Eisenberg et al.; Godson; Schaller). The replication of more complex phages such as λ and of the bacterial host itself have also been examined in some detail (Messer et al.; Hirota et al.; Wickner; Hobom et al.; Furth et al.; all this volume). However, our knowledge of how higher eukaryotic cells initiate and control replication lags far behind. This is due partly to the difficulties of studying complex genomes and partly to the lack of replication-defective mutants to guide us through the maze of enzymatic reactions, which must be associated with the process of replication
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