MafB is a transcription factor that induces myelomonocytic differentiation. However, the precise role of MafB in the pathogenic function of macrophages has never been clarified. Here we demonstrate ...that MafB promotes hyperlipidemic atherosclerosis by suppressing foam-cell apoptosis. Our data show that MafB is predominantly expressed in foam cells found within atherosclerotic lesions, where MafB mediates the oxidized LDL-activated LXR/RXR-induced expression of apoptosis inhibitor of macrophages (AIM). In the absence of MafB, activated LXR/RXR fails to induce the expression of AIM, a protein that is normally responsible for protecting macrophages from apoptosis; thus, Mafb-deficient macrophages are prone to apoptosis. Haematopoietic reconstitution with Mafb-deficient fetal liver cells in recipient LDL receptor-deficient hyperlipidemic mice revealed accelerated foam-cell apoptosis, which subsequently led to the attenuation of the early atherogenic lesion. These findings represent the first evidence that the macrophage-affiliated MafB transcription factor participates in the acceleration of atherogenesis.
c-Maf is one of the large Maf (musculoaponeurotic fibrosarcoma) transcription factors that belong to the activated protein-1 super family of basic leucine zipper proteins. Despite its overexpression ...in hematologic malignancies, the physiologic roles c-Maf plays in normal hematopoiesis have been largely unexplored. On a C57BL/6J background, c-Maf−/− embryos succumbed from severe erythropenia between embryonic day (E) 15 and E18. Flow cytometric analysis of fetal liver cells showed that the mature erythroid compartments were significantly reduced in c-Maf−/− embryos compared with c-Maf+/+ littermates. Interestingly, the CFU assay indicated there was no significant difference between c-Maf+/+ and c-Maf−/− fetal liver cells in erythroid colony counts. This result indicated that impaired definitive erythropoiesis in c-Maf−/− embryos is because of a non–cell-autonomous effect, suggesting a defective erythropoietic microenvironment in the fetal liver. As expected, the number of erythroblasts surrounding the macrophages in erythroblastic islands was significantly reduced in c-Maf−/− embryos. Moreover, decreased expression of VCAM-1 was observed in c-Maf−/− fetal liver macrophages. In conclusion, these results strongly suggest that c-Maf is crucial for definitive erythropoiesis in fetal liver, playing an important role in macrophages that constitute erythroblastic islands.
A survey was carried out to investigate the occurrence of
Sarcocystis infection in the loin (
Musculus longissimus) of Japanese and imported beef. In all, the muscle tissue of 482 samples were ...examined by histological method. The prevalence of
Sarcocystis unspecified species cysts was lower in Japanese beef (total 6.31%: 0% in Holstein castrated, 12.96% in Holstein milk cow, 3.33% in Japanese shorthorn and 11.58% in Japanese black cattle) than in beef imported from America (36.78%) or Australia (29.49%). The infection density of imported beef, especially in American, was higher than in Japanese beef. All detected cysts except one were identified as
Sarcocystis cruzi. One thick walled cyst was found in Australian beef but it could not be distinguished as either
Sarcocystis hirsuta or
Sarcocystis hominis.
Autophagy is a bulk degradation process that is conserved in eukaryotic cells and functions in the turnover of cytoplasmic materials and organelles. When eukaryotic cells face nutrient starvation, ...the autophagosome, a double‐membraned organelle, is generated from the pre‐autophagosomal structure (PAS). In the yeast Saccharomyces cerevisiae, 16 ATG (autophagy‐related) genes are essential for autophagosome formation. Most of the Atg proteins are involved in the PAS, leading to autophagosome production. However, the mechanism of PAS organization remains to be elucidated. Here, we performed a systematic and quantitative analysis by fluorescence microscopy to develop a hierarchy map of Atg proteins involved in PAS organization. This analysis suggests that Atg17p is the most basic protein in PAS organization: when it is specifically targeted to the plasma membrane, other Atg proteins are recruited to that location, suggesting that Atg17p acts as a scaffold protein to organize Atg proteins to the PAS.
Atg18 is essential for both autophagy and the regulation of vacuolar morphology. The latter process is mediated by phosphatidylinositol 3,5-bisphosphate binding, which is dispensable for autophagy. ...Atg18 also binds to phosphatidylinositol 3-phosphate (PtdIns(3)P) in vitro. Here, we investigate the relationship between PtdIns(3)P-binding of Atg18 and autophagy. Using an Atg18 variant, Atg18(FTTG), which is unable to bind phosphoinositides, we found that PtdIns(3)P binding of Atg18 is essential for full activity in both selective and nonselective autophagy. Atg18(FTTG) formed a complex with Atg2 in a normal manner, and Atg18-Atg2 complex formation occurred in cells in the absence of PtdIns(3)P, indicating that Atg18-Atg2 complex formation is independent of PtdIns(3)P-binding of Atg18. Atg18 localized to endosomes, the vacuolar membrane, and autophagic membranes, whereas Atg18(FTTG) did not localize to these structures. The localization of Atg2 to autophagic membranes was also lost in Atg18(FTTG) cells. These data indicate that PtdIns(3)P-binding of Atg18 is involved in directing the Atg18-Atg2 complex to autophagic membranes. Connection of a 2×FYVE domain, a specific PtdIns(3)P-binding domain, to the C terminus of Atg18(FTTG) restored the localization of Atg18-Atg2 to autophagic membranes and full autophagic activity, indicating that PtdIns(3)P-binding by Atg18 is dispensable for the function of the Atg18-Atg2 complex but is required for its localization. This also suggests that PtdIns(3)P does not act allosterically on Atg18. Taken together, Atg18 forms a complex with Atg2 irrespective of PtdIns(3)P binding, associates tightly to autophagic membranes by interacting with PtdIns(3)P, and plays an essential role.
During the process of autophagy, cytoplasmic materials are sequestered by double-membrane structures, the autophagosomes, and then transported to a lytic compartment to be degraded. One of the most ...fundamental questions about autophagy involves the origin of the autophagosomal membranes. In this study, we focus on the intracellular dynamics of Atg9, a multispanning membrane protein essential for autophagosome formation in yeast. We found that the vast majority of Atg9 existed on cytoplasmic mobile vesicles (designated Atg9 vesicles) that were derived from the Golgi apparatus in a process involving Atg23 and Atg27. We also found that only a few Atg9 vesicles were required for a single round of autophagosome formation. During starvation, several Atg9 vesicles assembled individually into the preautophagosomal structure, and eventually, they are incorporated into the autophagosomal outer membrane. Our findings provide conclusive linkage between the cytoplasmic Atg9 vesicles and autophagosomal membranes and offer new insight into the requirement for Atg9 vesicles at the early step of autophagosome formation.
Autophagy induced by nutrient depletion is involved in survival during starvation conditions. In addition to starvation-induced autophagy, the yeast Saccharomyces cerevisiae also has a constitutive ...autophagy-like system, the Cvt pathway. Among 31 autophagy-related (Atg) proteins, the function of Atg17, Atg29, and Atg31 is required specifically for autophagy. In this study, we investigated the role of autophagy-specific (i.e., non-Cvt) proteins under autophagy-inducing conditions. For this purpose, we used atg11Delta cells in which the Cvt pathway is abrogated. The autophagy-unique proteins are required for the localization of Atg proteins to the pre-autophagosomal structure (PAS), the putative site for autophagosome formation, under starvation condition. It is likely that these Atg proteins function as a ternary complex, because Atg29 and Atg31 bind to Atg17. The Atg1 kinase complex (Atg1-Atg13) is also essential for recruitment of Atg proteins to the PAS. The assembly of Atg proteins to the PAS is observed only under autophagy-inducing conditions, indicating that this structure is specifically involved in autophagosome formation. Our results suggest that Atg1 complex and the autophagy-unique Atg proteins cooperatively organize the PAS in response to starvation signals.
In the yeast Saccharomyces cerevisiae, two similar phosphatidylinositol 3-kinase complexes (complexes I and II) function in distinct biological processes, complex I in autophagy and complex II in the ...vacuolar protein sorting via endosomes. Atg14p is only integrated into complex I, likely facilitating the function of complex I in autophagy. Deletion analysis of Atg14p revealed that N-terminal region containing the coiled-coil structures was essential and sufficient for autophagy. Atg14p localized to pre-autophagosomal structure (PAS) and vacuolar membranes, whereas Vps38p, a component specific to complex II, localized to endosomes and vacuolar membranes. Vps34p and Vps30p, components shared by the two complexes, localized to the PAS, vacuolar membranes, and several punctate structures that included endosomes. The localization of these components to the PAS was Atg14p dependent but not dependent on Vps38p. Conversely, localization of these proteins to endosomes required Vps38p but not Atg14p. Vps15p, regulatory subunit of the Vps34p complexes, localized to the PAS, vacuolar membranes, and punctate structures independent of both Atg14p and Vps38p. Together, these results indicate that complexes I and II function in distinct biological processes by localizing to specific compartments in a manner mediated by specific components of each complex, Atg14p and Vps38p, respectively.
Amino acids stored in the vacuoles are exported to the cytosol mainly for protein synthesis; however, the molecular identity of vacuolar amino acid exporters remains obscure in plants. Here, we ...demonstrate that the heterologous expression of AtAVT3 genes, Arabidopsis homologs of AVT3 and AVT4 encoding vacuolar amino acid exporters in yeast, reduces vacuolar amino acid levels in the avt3∆avt4∆ yeast cells. In vitro experiments revealed that 14C‐labeled Ala and Pro are exported from vacuolar membrane vesicles by AtAvt3A in an ATP‐dependent manner. In Arabidopsis, AtAvt3A fused with green fluorescent protein localizes to the vacuolar membrane. We propose that AtAVT3 family represents the long sought‐for vacuolar amino acid exporters in plants.
Autophagy is a degradation system of cytoplasmic proteins and organelles via formation of double-membrane vesicles called autophagosomes. In the yeast Saccharomyces cerevisiae, autophagosomes are ...formed via the pre-autophagosomal structure (PAS) in a manner dependent on Atg proteins. Under nutrient-rich condition, Atg9 is recruited to the PAS by binding to Atg11 for the Cvt pathway. However, because Atg9 is recruited to the PAS in atg11Δ cells in starved condition and autophagy is induced, autophagy-specific mechanism for the Atg9 recruitment to the PAS has been assumed. Here, we demonstrate that, in autophagy-inducing condition, Atg9 is recruited to the PAS in a manner dependent on Atg17. Atg9 physically interacts with Atg17 in the presence of rapamycin. This interaction requires Atg1, a protein kinase essential for autophagy. Consistently, the Atg17-dependent PAS localization of Atg9 requires Atg1. However, its kinase activity is dispensable for this process. It rather regulates the equilibrium of assembly and disassembly of Atg9 at the PAS.
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