Kinsey Maundrell

Bcl-2 Undergoes Phosphorylation by c-Jun N-terminal Kinase/Stress-activated Protein Kinases in the Presence of the Constitutively Active GTP-binding Protein Rac1

We have studied the phosphorylation of the Bcl-2 family of proteins by different mitogen-activated protein (MAP) kinases. Purified Bcl-2 was found to be phosphorylated by the c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) p54-SAPKβ, and this is specific insofar as the extracellular signal-regulated kinase 1 (ERK1) and p38/RK/CSBP (p38) catalyzed only weak modification. Bcl-2 undergoes similar phosphorylation in COS-7 when coexpressed together with p54-SAPKβ and the constitutive Rac1 mutant G12V. This is seen by both 32PO4labeling and the appearance of five discrete Bcl-2 bands with reduced gel mobility. As anticipated, both intracellular p54-SAPKβ activation and Bcl-2 phosphorylation are blocked by co-transfection with the MAP kinase specific phosphatase MKP3/PYST1. MAP kinase specificity is also seen in COS-7 cells as Bcl-2 undergoes only weak phosphorylation when co-expressed with enzymatically activated ERK1 or p38. Four critical residues undergoing phosphorylation in COS-7 cells were identified by expression of the quadruple Bcl-2 point mutant T56A,S70A,T74A,S87A. Sequencing phosphopeptides derived from tryptic digests of Bcl-2 indicates that purified GST-p54-SAPKβ phosphorylates identical sitesin vitro. This is the first report of Bcl-2 phosphorylation by the JNK/SAPK class of MAP kinases and could indicate a key modification allowing control of Bcl-2 function by cell surface receptors, Rho family GTPases, and/or cellular stresses.

Mitochondrial RNA granules: Compartmentalizing mitochondrial gene expression

In mitochondria, DNA replication, gene expression, and RNA degradation machineries coexist within a common nondelimited space, raising the question of how functional compartmentalization of gene expression is achieved. Here, we discuss the recently characterized “mitochondrial RNA granules,” mitochondrial subdomains with an emerging role in the regulation of gene expression.

Reconstitution of caspase-mediated cell-death signalling in Schizosaccharomyces pombe

Abstract Two pro-apoptotic proteases, caspase-1 and caspase-3, have been expressed as full-length proteins in the fission yeast Schizosaccharomyces pombe. Both proteins autoprocess to generate the corresponding active enzyme and both are lethal to the yeast cell. Lethality is due to catalytic activity since the expression of the inactive mutant forms of both caspases does not result in an obvious phenotype. Caspase-expressing yeast can be rescued by co-expression of the baculovirus protein p35, a known inhibitor of the caspase family. Co-expression of Bcl-2, another anti-apoptotic protein, does not prevent the cell death induced by either caspase. However, Bcl-2 is itself cleaved by both caspase-1 and caspase-3 at two adjacent recognition sites, YEWD31′A and DAGD34′V respectively, immediately downstream from the N-terminal BH4 domain, a region of Bcl-2 which is essential for its anti-apoptotic activity; similar cleavage of Bcl-2 by caspases has been demonstrated in mammalian cells. Hence, key elements of the apoptotic pathway can be reliably reconstituted in fission yeast, opening the way to exploit yeast in order to study the control of apoptosis. Furthermore, the activity of caspase-3, although not caspase-1, can be demonstrated in vitro using chromogenic substrates. This offers the possibility of using caspase-producing strains of yeast to screen for chemical inhibitors either in vivo or in vitro.

Life without the mitochondrial calcium uniporter.

Calcium enters mitochondria through a dedicated channel referred to as the mitochondrial calcium uniporter (MCU), whose molecular identity has long remained elusive. Since the discovery of the gene encoding the MCU protein two years ago, researchers have awaited the generation of a mouse lacking the MCU. These mice are fully viable and show defects limited to performance of high-energy-demanding exercises. Strikingly, no protection against necrosis is observed following ischaemia-reperfusion in the heart.

The pseudouridine synthase RPUSD4 is an essential component of mitochondrial RNA granules

Mitochondrial gene expression is a fundamental process that is largely dependent on nuclear-encoded proteins. Several steps of mitochondrial RNA processing and maturation, including RNA post-transcriptional modification, appear to be spatially organized into distinct foci, which we have previously termed mitochondrial RNA granules (MRGs). Although an increasing number of proteins have been localized to MRGs, a comprehensive analysis of the proteome of these structures is still lacking. Here, we have applied a microscopy-based approach that has allowed us to identify novel components of the MRG proteome. Among these, we have focused our attention on RPUSD4, an uncharacterized mitochondrial putative pseudouridine synthase. We show that RPUSD4 depletion leads to a severe reduction of the steady-state level of the 16S mitochondrial (mt) rRNA with defects in the biogenesis of the mitoribosome large subunit and consequently in mitochondrial translation. We report that RPUSD4 binds 16S mt-rRNA, mt-tRNAMet, and mt-tRNAPhe, and we demonstrate that it is responsible for pseudouridylation of the latter. These data provide new insights into the relevance of RNA pseudouridylation in mitochondrial gene expression.

FASTKD1 and FASTKD4 have opposite effects on expression of specific mitochondrial RNAs, depending upon their endonuclease-like RAP domain

Abstract FASTK family proteins have been identified as regulators of mitochondrial RNA homeostasis linked to mitochondrial diseases, but much remains unknown about these proteins. We show that CRISPR-mediated disruption of FASTKD1 increases ND3 mRNA level, while disruption of FASTKD4 reduces the level of ND3 and of other mature mRNAs including ND5 and CYB, and causes accumulation of ND5–CYB precursor RNA. Disrupting both FASTKD1 and FASTKD4 in the same cell results in decreased ND3 mRNA similar to the effect of depleting FASTKD4 alone, indicating that FASTKD4 loss is epistatic. Interestingly, very low levels of FASTKD4 are sufficient to prevent ND3 loss and ND5–CYB precursor accumulation, suggesting that FASTKD4 may act catalytically. Furthermore, structural modeling predicts that each RAP domain of FASTK proteins contains a nuclease fold with a conserved aspartate residue at the putative active site. Accordingly, mutation of this residue in FASTKD4 abolishes its function. Experiments with FASTK chimeras indicate that the RAP domain is essential for the function of the FASTK proteins, while the region upstream determines RNA targeting and protein localization. In conclusion, this paper identifies new aspects of FASTK protein biology and suggests that the RAP domain function depends on an intrinsic nucleolytic activity.