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The ability of a neuron to regenerate its axon after injury depends in part on its intrinsic regenerative potential. Here, we identify novel intrinsic regulators of axon regeneration: poly(ADP-ribose) glycohodrolases (PARGs) and poly(ADP-ribose) polymerases (PARPs). PARGs, which remove poly(ADP-ribose) from proteins, act in injured GABA motor neurons to enhance axon regeneration. PARG expression is regulated by DLK signaling, and PARGs mediate DLK function in enhancing axon regeneration. Conversely, PARPs, which add poly(ADP-ribose) to proteins, inhibit axon regeneration of both GABA neurons and mammalian cortical neurons. Furthermore, chemical PARP inhibitors improve axon regeneration when administered after injury. Our results indicate that regulation of poly(ADP-ribose) levels is a critical function of the DLK regeneration pathway, that poly-(ADP ribosylation) inhibits axon regeneration across species, and that chemical inhibition of PARPs can elicit axon regeneration.
Fossil records indicate that life appeared in marine environments ∼3.5 billion years ago (Gyr) and transitioned to terrestrial ecosystems nearly 2.5 Gyr. Sequence analysis suggests that "hydrobacteria" and "terrabacteria" might have diverged as early as 3 Gyr. Bacteria of the genus Azospirillum are associated with roots of terrestrial plants; however, virtually all their close relatives are aquatic. We obtained genome sequences of two Azospirillum species and analyzed their gene origins. While most Azospirillum house-keeping genes have orthologs in its close aquatic relatives, this lineage has obtained nearly half of its genome from terrestrial organisms. The majority of genes encoding functions critical for association with plants are among horizontally transferred genes. Our results show that transition of some aquatic bacteria to terrestrial habitats occurred much later than the suggested initial divergence of hydro- and terrabacterial clades. The birth of the genus Azospirillum approximately coincided with the emergence of vascular plants on land.
Cell separation in Schizosaccharomyces pombe is achieved by the concerted action of the Eng1 endo-beta-1,3-glucanase and the Agn1 endo-alpha-1,3-glucanase, which are transported to the septum and localize to a ringlike structure that surrounds the septum. The requirements for the correct localization of both hydrolases as a ring were analyzed using green fluorescent protein fusion proteins. Targeting to the septum required a functional exocyst, because both proteins failed to localize correctly in sec8-1 or exo70delta mutants, suggesting that Agn1 and Eng1 might be two of the cargo proteins present in the vesicles that accumulate in exocyst mutants. Septins and Mid2 were also required for correct formation of a ring. In their absence, Eng1 and Agn1 were found in a disk-like structure that spanned the septum, rather than in a ring. Even though septin and mid2delta mutants have a cell separation defect, the septum and the distribution of linear beta-1,3-glucans were normal in these cells, suggesting that mislocalization of Eng1 and Agn1 might be the reason underlying the failure to separate efficiently. Thus, one of the functions of the septin ring would be to act as a positional marker for the localization of hydrolytic proteins to the medial region.
Poly(ADP-ribose) polymerase-1 (PARP-1) influences numerous cellular processes, including DNA repair, transcriptional regulation, and caspase-independent cell death, by utilizing NAD(+) to synthesize long chains of poly(ADP-ribose) (PAR) on target proteins, including itself. During the apoptotic response, caspases-3 and -7 cleave PARP-1, thereby inhibiting its activity. Here, we have examined the role of PARP-1 activation and cleavage in the latter stages of apoptosis in response to DNA fragmentation. PARP-1 poly(ADP-ribosyl)ation correlated directly with induction of apoptosis by the lipid peroxidation product, 4-hydroxy-2-nonenal. A significant decrease in PAR accumulation was observed upon caspase or DNA fragmentation factor 40 (DFF40) inhibition. Because DNA fragmentation mediated by DFF40 augmented PARP-1 modification status in apoptotic cells, we hypothesized that PARP-1 alters DFF40 function following PAR accumulation. Indeed, PARP-1, in the presence of NAD(+), significantly decreased DFF40 activity on plasmid substrates. Conversely, PARP-1 enhanced the DNase activity of DFF40 in the absence of NAD(+). The inhibition of DFF40 activity in the presence of NAD(+) was reduced by co-incubation with poly(ADP-ribose) glycohydrolase and a PARP inhibitor. Additionally, caspase-cleaved PARP-1, in the presence of NAD(+), did not inhibit DFF40 activity significantly. Our results suggest that PARP-1 poly(ADP-ribosyl)ation is a terminal event in the apoptotic response that occurs in response to DNA fragmentation and directly influences DFF40 activity.
The Saccharomyces cerevisiae genome contains five genes encoding P-type ATPases that are potential aminophospholipid translocases (APTs): DRS2, NEO1, and three uncharacterized open reading frames that we have named DNF1, DNF2, and DNF3 for DRS2/NEO1 family. NEO1 is the only essential gene in APT family and seems to be functionally distinct from the DRS2/DNF genes. The drs2Delta dnf1Delta dnf2Delta dnf3Delta quadruple mutant is inviable, although any one member of this group can maintain viability, indicating that there is a substantial functional overlap between the encoded proteins. We have previously implicated Drs2p in clathrin function at the trans-Golgi network. In this study, we constructed strains carrying all possible viable combinations of null alleles from this group and analyzed them for defects in protein transport. The drs2Delta dnf1Delta mutant grows slowly, massively accumulates intracellular membranes, and exhibits a substantial defect in the transport of alkaline phosphatase to the vacuole. Transport of carboxypeptidase Y to the vacuole is also perturbed, but to a lesser extent. In addition, the dnf1Delta dnf2Delta dnf3Delta mutant exhibits a defect in recycling of GFP-Snc1p in the early endocytic-late secretory pathways. Drs2p and Dnf3p colocalize with the trans-Golgi network marker Kex2p, whereas Dnf1p and Dnf2p seem to localize to the plasma membrane and late exocytic or early endocytic membranes. We propose that eukaryotes express multiple APT subfamily members to facilitate protein transport in multiple pathways.
BACKGROUND - Nuclear pore complexes (NPCs) are essential for facilitated, directional nuclear transport; however, the mechanism by which ~30 different nucleoporins (nups) are assembled into NPCs is unknown. We combined a genetic strategy in Saccharomyces cerevisiae with Green Fluorescence Protein (GFP) technology to identify mutants in NPC structure, assembly, and localization. To identify such mutants, a bank of temperature sensitive strains was generated and examined by fluorescence microscopy for mislocalization of GFP-tagged nups at the non-permissive temperature.
RESULTS - A total of 121 mutant strains were isolated, with most showing GFP-Nic96 and Nup170-GFP mislocalized to discrete, cytoplasmic foci. By electron microscopy, several mutants also displayed an expansion of the endoplasmic reticulum (ER). Complementation analysis identified several mutant groups with defects in components required for ER/Golgi trafficking (sec13, sec23, sec27, and bet3). By directed testing, we found that mutant alleles of all COPII components resulted in altered GFP-Nup localization. Finally, at least nine unknown complementation groups were identified that lack secretion defects.
CONCLUSION - The isolation of sec mutants in the screen could reflect a direct role for vesicle fusion or the COPII coat during NPC assembly; however, only those sec mutants that altered ER structure affected Nup localization. This suggests that the GFP-Nup mislocalization phenotypes observed in these mutants were the indirect result of overproliferation of the ER and connected outer nuclear envelope. The identification of potentially novel mutants with no secretory defects suggests the distinct GFP-Nup localization defects in other mutants in the collection will provide insights into NPC structure and assembly.
This study identified prostaglandin D2 synthase (PGDS) in murine epididymal fluid using a proteomic approach combining two-dimensional (2D) gel electrophoresis and mass spectrometry (MS). The caudal epididymal fluid was collected by retroperfusion, and proteins were separated by 2D gel electrophoresis followed by matrix-assisted laser desorption ionization MS analyses after trypsin digestion. The identification was based on the protein-specific peptide map as well as on sequence information generated by nano-electrospray ionization MS/MS. By in situ hybridization, the mRNA was detected in caput, corpus, and cauda, but it was not detected in the initial segment. The PGDS protein was mostly detected in the corpus and cauda by Western blot analysis and immunohistochemistry using a specific polyclonal antibody. In caudal fluid, PGDS was distributed among several isoforms (pI range, 6.5-8.8), suggesting that this protein undergoes posttranslational modification of its primary sequence. After N-glycanase digestion, the molecular mass decreased from 20-25 to 18.5 kDa, its theoretical mass. The PGDS was also detected in the epididymis of rat, hamster, and cynomolgus monkey from the caput to the cauda. In conclusion, MS is a powerful and accurate technique that allows unambiguous identification of the murine epididymal PGDS. The protein is 1) present throughout the epididymis, except in the initial segment, with an increasing luminal concentration from distal caput to cauda; 2) a major protein in caudal fluid; 3) an N-glycosylated, highly polymorphic protein; and 4) conserved during evolution.
ADP-ribosylation factor (ARF) is a small GTP-binding protein that is thought to regulate the assembly of coat proteins on transport vesicles. To identify factors that functionally interact with ARF, we have performed a genetic screen in Saccharomyces cerevisiae for mutations that exhibit synthetic lethality with an arf1Delta allele and defined seven genes by complementation tests (SWA1-7 for synthetically lethal with arf1Delta). Most of the swa mutants exhibit phenotypes comparable to arf1Delta mutants such as temperature-conditional growth, hypersensitivity to fluoride ions, and partial protein transport and glycosylation defects. Here, we report that swa5-1 is a new temperature-sensitive allele of the clathrin heavy chain gene (chc1-5), which carries a frameshift mutation near the 3' end of the CHC1 open reading frame. This genetic interaction between arf1 and chc1 provides in vivo evidence for a role for ARF in clathrin coat assembly. Surprisingly, strains harboring chc1-5 exhibited a significant defect in transport of carboxypeptidase Y or carboxypeptidase S to the vacuole that was not observed in other chc1 ts mutants. The kinetics of invertase secretion or transport of alkaline phosphatase to the vacuole were not significantly affected in the chc1-5 mutant, further implicating clathrin specifically in the Golgi to vacuole transport pathway for carboxypeptidase Y.
alpha 1,3 mannosyltransferase (Mnn1p) is a type II integral membrane protein that is localized to the yeast Golgi complex. We have examined the signals within Mnn1p that mediate Golgi localization by expression of fusion proteins comprised of Mnn1p and the secreted protein invertase. The N-terminal transmembrane domain (TMD) of Mnn1p is sufficient to localize invertase to the Golgi complex by a mechanism that is not saturable by approximately 15-20 fold overexpression. Furthermore, the TMD-mediated localization mechanism is clathrin dependent, as an invertase fusion protein bearing only the Mnn1p TMD is mislocalized to the plasma membrane of a clathrin heavy chain mutant. The Mnn1-invertase fusion proteins are not retained in the Golgi complex as efficiently as Mnn1p, suggesting that other signals may be present in the wild-type protein. Indeed, the Mnn1p lumenal domain (Mnn1-s) is also localized to the Golgi complex when expressed as a functional, soluble protein by exchanging its TMD for a cleavable signal sequence. In contrast to the Mnn1-invertase fusion proteins, overexpression of Mnn1-s saturates its retention mechanism, and results in the partial secretion of this protein. These data indicate that Mnn1p has separable Golgi localization signals within both its transmembrane and lumenal domains.