Roles for a lipid phosphatase in the activation of its opposing lipid kinase.

Fig4 is a phosphoinositide phosphatase that converts PI3,5P2 to PI3P. Paradoxically, mutation of Fig4 results in lower PI3,5P2, indicating that Fig4 is also required for PI3,5P2 production. Fig4 promotes elevation of PI3,5P2, in part, through stabilization of a protein complex that includes its opposing lipid kinase, Fab1, and the scaffold protein Vac14. Here we show that multiple regions of Fig4 contribute to its roles in the elevation of PI3,5P2: its catalytic site, an N-terminal disease-related surface, and a C-terminal region. We show that mutation of the Fig4 catalytic site enhances the formation of the Fab1-Vac14-Fig4 complex, and reduces the ability to elevate PI3,5P2. This suggests that independent of its lipid phosphatase function, the active site plays a role in the Fab1-Vac14-Fig4 complex. We also show that the N-terminal disease-related surface contributes to the elevation of PI3,5P2 and promotes Fig4 association with Vac14 in a manner that requires the Fig4 C-terminus. We find that the Fig4 C-terminus alone interacts with Vac14 in vivo and retains some functions of full-length Fig4. Thus, a subset of Fig4 functions are independent of its phosphatase domain and at least three regions of Fig4 play roles in the function of the Fab1-Vac14-Fig4 complex.

The ATPase Fap7 Tests the Ability to Carry Out Translocation-like Conformational Changes and Releases Dim1 during 40S Ribosome Maturation.

Late in their maturation, nascent small (40S) ribosomal subunits bind 60S subunits to produce 80S-like ribosomes. Because of the analogy of this translation-like cycle to actual translation, and because 80S-like ribosomes do not produce any protein, it has been suggested that this represents a quality control mechanism for subunit functionality. Here we use genetic and biochemical experiments to show that the essential ATPase Fap7 promotes formation of the rotated state, a key intermediate in translocation, thereby releasing the essential assembly factor Dim1 from pre-40S subunits. Bypassing this quality control step produces defects in reading frame maintenance. These results show how progress in the maturation cascade is linked to a test for a key functionality of 40S ribosomes: their ability to translocate the mRNA⋅tRNA pair. Furthermore, our data demonstrate for the first time that the translation-like cycle is a quality control mechanism that ensures the fidelity of the cellular ribosome pool.

PI5P and PI(3,5)P2: Minor, but Essential Phosphoinositides.

In most eukaryotes, phosphoinositides (PIs) have crucial roles in multiple cellular functions. Although the cellular levels of phosphatidylinositol 5-phosphate (PI5P) and phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2) are extremely low relative to some other PIs, emerging evidence demonstrates that both lipids are crucial for the endocytic pathway, intracellular signaling, and adaptation to stress. Mutations that causes defects in the biosynthesis of PI5P and PI(3,5)P2 are linked to human diseases including neurodegenerative disorders. Here, we review recent findings on cellular roles of PI5P and PI(3,5)P2, as well as the pathophysiological importance of these lipids.Key words: Phosphoinositides, Membrane trafficking, Endocytosis, Vacuoles/Lysosomes, Fab1/PIKfyve.

An intramolecular interaction within the lipid kinase Fab1 regulates cellular phosphatidylinositol 3,5-bisphosphate lipid levels.

Phosphorylated phosphoinositide lipids (PPIs) are low-abundance signaling molecules that control signal transduction pathways and are necessary for cellular homeostasis. The PPI phosphatidylinositol (3,5)-bisphosphate (PI(3,5)P2) is essential in multiple organ systems. PI(3,5)P2 is generated from PI3P by the conserved lipid kinase Fab1/PIKfyve. Defects in the dynamic regulation of PI(3,5)P2 are linked to human diseases. However, few mechanisms that regulate PI(3,5)P2 have been identified. Here we report an intramolecular interaction between the yeast Fab1 kinase region and an upstream conserved cysteine-rich (CCR) domain. We identify mutations in the kinase domain that lead to elevated levels of PI(3,5)P2 and impair the interaction between the kinase and CCR domain. We also identify mutations in the CCR domain that lead to elevated levels of PI(3,5)P2 Together these findings reveal a regulatory mechanism that involves the CCR domain of Fab1 and contributes to dynamic control of cellular PI(3,5)P2 synthesis.

Close encounters of the lysosome-peroxisome kind.

Lysosomes provide a major source for cellular cholesterol; however, most of this cholesterol is trafficked to the plasma membrane via unknown mechanisms. Chu et al. identify an unexpected role for peroxisomes in the transport of cholesterol from the lysosome to the plasma membrane via a lysosome-peroxisome membrane contact site.

Activity-dependent PI(3,5)P2 synthesis controls AMPA receptor trafficking during synaptic depression.

Dynamic regulation of phosphoinositide lipids (PIPs) is crucial for diverse cellular functions, and, in neurons, PIPs regulate membrane trafficking events that control synapse function. Neurons are particularly sensitive to the levels of the low abundant PIP, phosphatidylinositol 3,5-bisphosphate [PI(3,5)P2], because mutations in PI(3,5)P2-related genes are implicated in multiple neurological disorders, including epilepsy, severe neuropathy, and neurodegeneration. Despite the importance of PI(3,5)P2 for neural function, surprisingly little is known about this signaling lipid in neurons, or any cell type. Notably, the mammalian homolog of yeast vacuole segregation mutant (Vac14), a scaffold for the PI(3,5)P2 synthesis complex, is concentrated at excitatory synapses, suggesting a potential role for PI(3,5)P2 in controlling synapse function and/or plasticity. PI(3,5)P2 is generated from phosphatidylinositol 3-phosphate (PI3P) by the lipid kinase PI3P 5-kinase (PIKfyve). Here, we present methods to measure and control PI(3,5)P2 synthesis in hippocampal neurons and show that changes in neural activity dynamically regulate the levels of multiple PIPs, with PI(3,5)P2 being among the most dynamic. The levels of PI(3,5)P2 in neurons increased during two distinct forms of synaptic depression, and inhibition of PIKfyve activity prevented or reversed induction of synaptic weakening. Moreover, altering neuronal PI(3,5)P2 levels was sufficient to regulate synaptic strength bidirectionally, with enhanced synaptic function accompanying loss of PI(3,5)P2 and reduced synaptic strength following increased PI(3,5)P2 levels. Finally, inhibiting PI(3,5)P2 synthesis alters endocytosis and recycling of AMPA-type glutamate receptors (AMPARs), implicating PI(3,5)P2 dynamics in AMPAR trafficking. Together, these data identify PI(3,5)P2-dependent signaling as a regulatory pathway that is critical for activity-dependent changes in synapse strength.

A translation-like cycle is a quality control checkpoint for maturing 40S ribosome subunits.

Assembly factors (AFs) prevent premature translation initiation on small (40S) ribosomal subunit assembly intermediates by blocking ligand binding. However, it is unclear how AFs are displaced from maturing 40S ribosomes, if or how maturing subunits are assessed for fidelity, and what prevents premature translation initiation once AFs dissociate. Here we show that maturation involves a translation-like cycle whereby the translation factor eIF5B, a GTPase, promotes joining of large (60S) subunits with pre-40S subunits to give 80S-like complexes, which are subsequently disassembled by the termination factor Rli1, an ATPase. The AFs Tsr1 and Rio2 block the mRNA channel and initiator tRNA binding site, and therefore 80S-like ribosomes lack mRNA or initiator tRNA. After Tsr1 and Rio2 dissociate from 80S-like complexes Rli1-directed displacement of 60S subunits allows for translation initiation. This cycle thus provides a functional test of 60S subunit binding and the GTPase site before ribosomes enter the translating pool.

Ribosome assembly factors prevent premature translation initiation by 40S assembly intermediates.

Ribosome assembly in eukaryotes requires approximately 200 essential assembly factors (AFs) and occurs through ordered events that initiate in the nucleolus and culminate in the cytoplasm. Here, we present the electron cryo-microscopy (cryo-EM) structure of a late cytoplasmic 40S ribosome assembly intermediate from Saccharomyces cerevisiae at 18 angstrom resolution. We obtained cryo-EM reconstructions of preribosomal complexes lacking individual components to define the positions of all seven AFs bound to this intermediate. These late-binding AFs are positioned to prevent each step in the translation initiation pathway. Together, they obstruct the binding sites for initiation factors, prevent the opening of the messenger RNA channel, block 60S subunit joining, and disrupt the decoding site. These redundant mechanisms probably ensure that pre-40S particles do not enter the translation pathway, which would result in their rapid degradation.

SET7/9 catalytic mutants reveal the role of active site water molecules in lysine multiple methylation.

SET domain lysine methyltransferases (KMTs) methylate specific lysine residues in histone and non-histone substrates. These enzymes also display product specificity by catalyzing distinct degrees of methylation of the lysine ε-amino group. To elucidate the molecular mechanism underlying this specificity, we have characterized the Y245A and Y305F mutants of the human KMT SET7/9 (also known as KMT7) that alter its product specificity from a monomethyltransferase to a di- and a trimethyltransferase, respectively. Crystal structures of these mutants in complex with peptides bearing unmodified, mono-, di-, and trimethylated lysines illustrate the roles of active site water molecules in aligning the lysine ε-amino group for methyl transfer with S-adenosylmethionine. Displacement or dissociation of these solvent molecules enlarges the diameter of the active site, accommodating the increasing size of the methylated ε-amino group during successive methyl transfer reactions. Together, these results furnish new insights into the roles of active site water molecules in modulating lysine multiple methylation by SET domain KMTs and provide the first molecular snapshots of the mono-, di-, and trimethyl transfer reactions catalyzed by these enzymes.

Loss of the SIN3 transcriptional corepressor results in aberrant mitochondrial function.

BACKGROUND: SIN3 is a transcriptional repressor protein known to regulate many genes, including a number of those that encode mitochondrial components. RESULTS: By monitoring RNA levels, we find that loss of SIN3 in Drosophila cultured cells results in up-regulation of not only nuclear encoded mitochondrial genes, but also those encoded by the mitochondrial genome. The up-regulation of gene expression is accompanied by a perturbation in ATP levels in SIN3-deficient cells, suggesting that the changes in mitochondrial gene expression result in altered mitochondrial activity. In support of the hypothesis that SIN3 is necessary for normal mitochondrial function, yeast sin3 null mutants exhibit very poor growth on non-fermentable carbon sources and show lower levels of ATP and reduced respiration rates. CONCLUSIONS: The findings that both yeast and Drosophila SIN3 affect mitochondrial activity suggest an evolutionarily conserved role for SIN3 in the control of cellular energy production.

Powering through ribosome assembly.

Ribosome assembly is required for cell growth in all organisms. Classic in vitro work in bacteria has led to a detailed understanding of the biophysical, thermodynamic, and structural basis for the ordered and correct assembly of ribosomal proteins on ribosomal RNA. Furthermore, it has enabled reconstitution of active subunits from ribosomal RNA and proteins in vitro. Nevertheless, recent work has shown that eukaryotic ribosome assembly requires a large macromolecular machinery in vivo. Many of these assembly factors such as ATPases, GTPases, and kinases hydrolyze nucleotide triphosphates. Because these enzymes are likely regulatory proteins, much work to date has focused on understanding their role in the assembly process. Here, we review these factors, as well as other sources of energy, and their roles in the ribosome assembly process. In addition, we propose roles of energy-releasing enzymes in the assembly process, to explain why energy is used for a process that occurs largely spontaneously in bacteria. Finally, we use literature data to suggest testable models for how these enzymes could be used as targets for regulation of ribosome assembly.