Mak5 and Ebp2 act together on early pre-60S particles and their reduced functionality bypasses the requirement for the essential pre-60S factor Nsa1.

Ribosomes are the molecular machines that translate mRNAs into proteins. The synthesis of ribosomes is therefore a fundamental cellular process and consists in the ordered assembly of 79 ribosomal proteins (r-proteins) and four ribosomal RNAs (rRNAs) into a small 40S and a large 60S ribosomal subunit that form the translating 80S ribosomes. Most of our knowledge concerning this dynamic multi-step process comes from studies with the yeast Saccharomyces cerevisiae, which have shown that assembly and maturation of pre-ribosomal particles, as they travel from the nucleolus to the cytoplasm, relies on a multitude (>200) of biogenesis factors. Amongst these are many energy-consuming enzymes, including 19 ATP-dependent RNA helicases and three AAA-ATPases. We have previously shown that the AAA-ATPase Rix7 promotes the release of the essential biogenesis factor Nsa1 from late nucleolar pre-60S particles. Here we show that mutant alleles of genes encoding the DEAD-box RNA helicase Mak5, the C/D-box snoRNP component Nop1 and the rRNA-binding protein Nop4 bypass the requirement for Nsa1. Interestingly, dominant-negative alleles of RIX7 retain their phenotype in the absence of Nsa1, suggesting that Rix7 may have additional nuclear substrates besides Nsa1. Mak5 is associated with the Nsa1 pre-60S particle and synthetic lethal screens with mak5 alleles identified the r-protein Rpl14 and the 60S biogenesis factors Ebp2, Nop16 and Rpf1, which are genetically linked amongst each other. We propose that these 'Mak5 cluster' factors orchestrate the structural arrangement of a eukaryote-specific 60S subunit surface composed of Rpl6, Rpl14 and Rpl16 and rRNA expansion segments ES7L and ES39L. Finally, over-expression of Rix7 negatively affects growth of mak5 and ebp2 mutant cells both in the absence and presence of Nsa1, suggesting that Rix7, at least when excessively abundant, may act on structurally defective pre-60S subunits and may subject these to degradation.

Synchronizing nuclear import of ribosomal proteins with ribosome assembly.

Ribosomal proteins are synthesized in the cytoplasm, before nuclear import and assembly with ribosomal RNA (rRNA). Little is known about coordination of nucleocytoplasmic transport with ribosome assembly. Here, we identify a transport adaptor, symportin 1 (Syo1), that facilitates synchronized coimport of the two 5S-rRNA binding proteins Rpl5 and Rpl11. In vitro studies revealed that Syo1 concomitantly binds Rpl5-Rpl11 and furthermore recruits the import receptor Kap104. The Syo1-Rpl5-Rpl11 import complex is released from Kap104 by RanGTP and can be directly transferred onto the 5S rRNA. Syo1 can shuttle back to the cytoplasm by interaction with phenylalanine-glycine nucleoporins. X-ray crystallography uncovered how the α-solenoid symportin accommodates the Rpl5 amino terminus, normally bound to 5S rRNA, in an extended groove. Symportin-mediated coimport of Rpl5-Rpl11 could ensure coordinated and stoichiometric incorporation of these proteins into pre-60S ribosomes.

Up-regulation of hepatic lipolysis stimulated lipoprotein receptor by leptin: a potential lever for controlling lipid clearance during the postprandial phase.

As a hepatic receptor for triglyceride-rich lipoproteins, the lipolysis-stimulated lipoprotein receptor (LSR) may be involved in the dynamics of lipid distribution between the liver and peripheral tissues. Here, we explore the potential role of leptin in regulating LSR. At physiological concentrations (1-10 ng/ml), leptin increased LSR protein and mRNA levels in Hepa1-6 cells through an ERK1/2-dependent and α-amanitin-sensitive pathway. In vivo, leptin treatment of C57BL6/Rj mice (1 µg 2×/d, 8 d) led to a significant increase in hepatic LSR mRNA and protein, decreased liver triglycerides and increased VLDL secretion as compared to controls. LSR(+/-) mice with elevated postprandial lipemia placed on a high-fat (60% kcal) diet exhibited accelerated weight gain and increased fat mass as compared to controls. While plasma leptin levels were increased 3-fold, hepatic leptin receptor protein levels and phosphorylation of ERK1/2 were significantly reduced. Therefore, leptin is an important regulator of LSR protein levels providing the means for the control of hepatic uptake of lipids during the postprandial phase. However, this may no longer be functional in LSR(+/-) mice placed under a chronic dietary fat load, suggesting that this animal model could be useful for the study of molecular mechanisms involved in peripheral leptin resistance.

Oligomeric Dop1p is part of the endosomal Neo1p-Ysl2p-Arl1p membrane remodeling complex.

Yeast Dop1p is an essential protein that is highly conserved in evolution and whose function is largely unknown. Here, we provide evidence that Dop1p localizes to endosomes and exists in a complex with two other conserved proteins: Neo1p, a P(4)-ATPase and putative flippase, and the scaffolding protein Ysl2p/Mon2p. The latter operates during membrane budding at the tubular endosomal network/trans-Golgi network (TEN/TGN) in a process that includes clathrin recruitment via adaptor proteins. Consistent with a role for Dop1p during this process, temperature-sensitive dop1-3 cells accumulate multivesicular, elongated tubular and ring-like structures similar to those displayed by neo1 and ysl2 mutants. In further agreement with the concept of Dop1p-Neo1p-Ysl2p complex formation and co-operation, we show that dop1-3 cells exhibit reduced levels of Neo1p and Ysl2p at steady state. Conversely, mutations or deletions in NEO1 and YSL2 lead to a decrease in Dop1p levels. In addition to binding to Neo1p and Ysl2p, Dop1p can form dimers or multimers. A critical region for dimerization resides in the C-terminus with leucine zipper-like domains. Dop1p's membrane association is largely mediated by its internal region, but Ysl2p might not be crucial for membrane recruitment.

Lipolysis stimulated lipoprotein receptor: a novel molecular link between hyperlipidemia, weight gain, and atherosclerosis in mice.

The lipolysis-stimulated lipoprotein receptor, LSR, is a multimeric protein complex in the liver that undergoes conformational changes upon binding of free fatty acids, thereby revealing a binding site (s) that recognizes both apoB and apoE. Complete inactivation of the LSR gene is embryonic lethal in mice. Here we show that removal of a single LSR allele (LSR(-/+)) caused statistically significant increases in both plasma triglyceride and cholesterol levels, a 2-fold increase in plasma triglyceride changes during the post-prandial phase, and delayed clearance of lipid emulsions or a high fat meal. The longer postprandial lipoprotein clearance time observed in LSR(-/+) mice was further increased in LSR(-/+) mice lacking functional low density lipoprotein (LDL) receptors. LSR(-/+) mice placed on a Western-type diet displayed higher plasma triglycerides and cholesterol levels, increased triglyceride-rich lipoproteins and LDL, and increased aorta lipid content, as compared with control mice on the same diet. Furthermore, a direct correlation was observed between the hyperlipidemia and weight gain but only in the LSR(-/+) mice. Knockdown of LSR expression by small interfering RNA in mouse Hepa1-6 cells led to decreased internalization of both DiI-labeled cyclohexanedione-LDL and very low density lipoprotein in the presence of oleate. These data led us to conclude that LSR contributes to the physiological clearance of atherogenic triglyceride-rich lipoproteins and LDL. We propose that LSR cooperates with the LDL receptor in the final hepatic processing of apoB-containing lipoproteins and represents a novel therapeutic target for the treatment of hyperlipidemia associated with obesity and atherosclerosis.