Pioneering biochemical studies have long forged the concept that the mitochondria are the 'energy powerhouse of the cell'. These studies, combined with the unique evolutionary origin of the mitochondria, led the way to decades of research focusing on the organelle as an essential, yet independent, functional component of the cell. Recently, however, our conceptual view of this isolated organelle has been profoundly altered with the discovery that mitochondria function within an integrated reticulum that is continually remodeled by both fusion and fission events. The identification of a number of proteins that regulate these activities is beginning to provide mechanistic details of mitochondrial membrane remodeling. However, the broader question remains regarding the underlying purpose of mitochondrial dynamics and the translation of these morphological transitions into altered functional output. One hypothesis has been that mitochondrial respiration and metabolism may be spatially and temporally regulated by the architecture and positioning of the organelle. Recent evidence supports and expands this idea by demonstrating that mitochondria are an integral part of multiple cell signaling cascades. Interestingly, proteins such as GTPases, kinases and phosphatases are involved in bi-directional communication between the mitochondrial reticulum and the rest of the cell. These proteins link mitochondrial function and dynamics to the regulation of metabolism, cell-cycle control, development, antiviral responses and cell death. In this review we will highlight the emerging evidence that provides molecular definition to mitochondria as a central platform in the execution of diverse cellular events.

Dynamin-related protein 1 (DRP1) plays an important role in mitochondrial fission at steady state and during apoptosis. Using fluorescence recovery after photobleaching, we demonstrate that in healthy cells, yellow fluorescent protein (YFP)–DRP1 recycles between the cytoplasm and mitochondria with a half-time of 50 s. Strikingly, during apoptotic cell death, YFP-DRP1 undergoes a transition from rapid recycling to stable membrane association. The rapid cycling phase that characterizes the early stages of apoptosis is independent of Bax/Bak. However, after Bax recruitment to the mitochondrial membranes but before the loss of mitochondrial membrane potential, YFP-DRP1 becomes locked on the membrane, resulting in undetectable fluorescence recovery. This second phase in DRP1 cycling is dependent on the presence of Bax/Bak but independent of hFis1 and mitochondrial fragmentation. Coincident with Bax activation, we detect a Bax/Bak-dependent stimulation of small ubiquitin-like modifier-1 conjugation to DRP1, a modification that correlates with the stable association of DRP1 with mitochondrial membranes. Altogether, these data demonstrate that the apoptotic machinery regulates the biochemical properties of DRP1 during cell death.

Intersectin-s is a modular scaffolding protein regulating the formation of clathrin-coated vesicles. In addition to the Eps15 homology (EH) and Src homology 3 (SH3) domains of intersectin-s, the neuronal variant (intersectin-l) also has Dbl homology (DH), pleckstrin homology (PH) and C2 domains. We now show that intersectin-l functions through its DH domain as a guanine nucleotide exchange factor (GEF) for Cdc42. In cultured cells, expression of DH-domain-containing constructs cause actin rearrangements specific for Cdc42 activation. Moreover, in vivo studies reveal that stimulation of Cdc42 by intersectin-l accelerates actin assembly via N-WASP and the Arp2/3 complex. N-WASP binds directly to intersectin-l and upregulates its GEF activity, thereby generating GTP-bound Cdc42, a critical activator of N-WASP. These studies reveal a role for intersectin-l in a novel mechanism of N-WASP activation and in regulation of the actin cytoskeleton.

Tandem MS has identified 209 proteins of clathrin-coated vesicles (CCVs) isolated from rat brain. An overwhelming abundance of peptides were assigned to the clathrin coat with a 1:1 stoichiometry observed for clathrin heavy and light chains and a 2:1 stoichiometry of clathrin heavy chain with clathrin adaptor protein heterotetramers. Thirty-two proteins representing many of the known components of synaptic vesicles (SVs) were identified, supporting that a main function for brain CCVs is to recapture SVs after exocytosis. A ratio of vesicle-N-ethylmaleimide-sensitive factor attachment protein receptors to target-N-ethylmaleimide-sensitive factor attachment protein receptors, similar to that previously detected on SVs, supports a single-step model for SV sorting during CCV-mediated recycling of SVs. The uncovering of eight previously undescribed proteins, four of which have to date been linked to clathrin-mediated trafficking, further attests to the value of the current organelle-based proteomics strategy. T he sorting of receptors and other cell-surface proteins from the plasma membrane via clathrin-mediated endocytosis is the basis for a range of essential cellular processes, including the uptake of nutrient and signaling receptors, the control of cell and serum homeostasis through the internalization of plasma membrane pumps, and a contribution to learning and memory through the regulation of surface expression of neurotransmitter receptors (1). Until recently, it was thought that clathrin assembly into progressively curved lattices provided the driving force for the formation of clathrin-coated pits (CCPs) and vesicles (CCVs), and that the adaptor protein 2 (AP-2) complex was solely responsible for recruiting clathrin to the membrane and for binding to endocytic cargo, concentrating the cargo in CCPs (1, 2). However, clathrin assembly may not be sufficient to drive membrane curvature (3), and the previously accepted obligatory role for AP-2 in coat assembly and cargo recruitment has been recently questioned (4-6).In neuronal tissues, CCVs are postulated to be responsible for the recycling of synaptic vesicles (SVs) during neurotransmission (7). As such, CCVs retrieve SV membranes from the plasma membrane after SV collapse, concomitant with neurotransmitter release. Many of the components of the endocytic machinery are concentrated in the presynaptic compartment (8), and disruption of these proteins affects neurotransmission (9). Moreover, a number of SV proteins have been identified as components of isolated CCVs (10, 11). Synaptic transmission involving intermittent fusion of SVs without complete collapse (12, 13) has also been demonstrated. The prevalence of such a ''kiss-and-run'' mechanism with the alternative model of full fusion is uncertain (14). Even in the membrane retrieval model via CCVs, it remains unclear whether SVs are generated directly from CCVs (15, 16) or whether they require an additional sorting step through endosomal membranes localized in the presynaptic compartment (7, 17). Here, using ...

In brain, mRNAs are transported from the cell body to the processes, allowing for local protein translation at sites distant from the nucleus. Using subcellular fractionation, we isolated a fraction from rat embryonic day 18 brains enriched for structures that resemble amorphous collections of ribosomes. This fraction was enriched for the mRNA encoding beta-actin, an mRNA that is transported in dendrites and axons of developing neurons. Abundant protein components of this fraction, determined by tandem mass spectrometry, include ribosomal proteins, RNA-binding proteins, microtubule-associated proteins (including the motor protein dynein), and several proteins described only as potential open reading frames. The conjunction of RNA-binding proteins, transported mRNA, ribosomal machinery, and transporting motor proteins defines these structures as RNA granules. Expression of a subset of the identified proteins in cultured hippocampal neurons confirmed that proteins identified in the proteomics were present in neurites associated with ribosomes and mRNAs. Moreover many of the expressed proteins co-localized together. Time lapse video microscopy indicated that complexes containing one of these proteins, the DEAD box 3 helicase, migrated in dendrites of hippocampal neurons at the same speed as that reported for RNA granules. Although the speed of the granules was unchanged by activity or the neurotrophin brain-derived neurotrophic factor, brain-derived neurotrophic factor, but not activity, increased the proportion of moving granules. These studies define the isolation and composition of RNA granules expressed in developing brain.

Increased synthesis of Apolipoprotein A-I (ApoA-I) and HDL is believed to provide a new approach to treating atherosclerosis through the stimulation of reverse cholesterol transport. RVX-208 increases the production of ApoA-I in hepatocytes in vitro, and in vivo in monkeys and humans, which results in increased HDL-C, but the molecular target was not previously reported. Using binding assays and X-ray crystallography, we now show that RVX-208 selectively binds to bromodomains of the BET (Bromodomain and Extra Terminal) family, competing for a site bound by the endogenous ligand, acetylated lysine, and that this accounts for its pharmacological activity. siRNA experiments further suggest that induction of ApoA-I mRNA is mediated by BET family member BRD4. These data indicate that RVX-208 increases ApoA-I production through an epigenetic mechanism and suggests that BET inhibition may be a promising new approach to the treatment of atherosclerosis.

IntroductionClathrin-mediated membrane budding drives the formation of endocytic vesicles for the internalization of nutrients, receptors and other proteins at the cell surface Conner and Schmid, 2003a). It also operates at the trans-Golgi network (TGN), where it mediates the trafficking of cargo proteins from the TGN to the endosomal/lysosomal system (Brodsky et al., 2001;Hinners and Tooze, 2003). Several studies have revealed the complex molecular machinery underlying the formation and function of clathrin-coated pits (CCPs) and vesicles (CCVs) at the plasma membrane (Slepnev and De Camilli, 2000;Takei and Haucke, 2001). By contrast, the mechanisms of clathrin-mediated events at the TGN remain less clear. Recently, two new and highly related modules, the epsin N-terminal homology (ENTH) and AP180 N-terminal homology (ANTH) domains, have been identified in proteins that participate in clathrin-mediated budding. In this Commentary, we describe recent structural studies that have significantly advanced our understanding of E/ANTH domains. We also discuss how the identification of new E/ANTH proteins at the TGN, notably the ENTH-domain-containing protein enthoprotin, provides evidence that E/ANTH domains are universal elements in the clathrin-budding machinery. Identification of the ENTH domainThe heterotetrameric clathrin adaptor protein-2 (AP-2) complex plays an important role in the formation of endocytic vesicles. AP-2 contributes to the recruitment of clathrin triskelia to the plasma membrane and the assembly of triskelia into clathrin coats, leading to the formation of CCPs that eventually pinch off from the membrane in a dynamindependent manner to form CCVs (Brodsky et al., 2001; Sever, 2002). AP-2 also binds to endocytic cargo, leading to the concentration of cargo in nascent CCPs (Brodsky et al., 2001). Additionally, AP-2 recruits a diverse array of endocytic accessory proteins to sites of CCV formation (Slepnev and De Camilli, 2000;Takei and Haucke, 2001). These proteins bind through short consensus peptide motifs to a globular domain at the C-terminus of the AP-2 α-adaptin subunit termed the α-ear (Owen et al., 1999;Traub et al., 1999;Brett et al., 2002;Ritter et al., 2003).Members of the epsin family of endocytic regulatory proteins are important binding partners for the α-ear (Chen et al., 1998;Yamabhai et al., 1998;Nakashima et al., 1999;Rosenthal et al., 1999;Spradling et al., 2001) (Fig. 1). The best characterized member of this family, epsin 1, is unusual in that the C-terminal two-thirds of the molecule contains essentially no secondary structure (Kalthoff et al., 2002a). Within this extended, unstructured domain are multiple short peptide motifs that mediate interactions with endocytic proteins (Fig. 1). Included are eight copies of the DPW tripeptide that mediates binding to the α-ear (Owen et al., 1999;Traub et al., 1999) and two distinct clathrin-binding motifs, which mediate interactions with the clathrin heavy chain (Hussain et al., 1999;Rosenthal et al., 1999;Drake and Traub, 2001). Epsin ...

We recently identified polynucleotide phosphorylase (PNPase) as a potential binding partner for the TCL1 oncoprotein. Mammalian PNPase exhibits exoribonuclease and poly(A) polymerase activities, and PNPase overexpression inhibits cell growth, induces apoptosis, and stimulates proinflammatory cytokine production. A physiologic connection for these anticancer effects and overexpression is difficult to reconcile with the presumed mitochondrial matrix localization for endogenous PNPase, prompting this study. Here we show that basal and interferon-␤-induced PNPase was efficiently imported into energized mitochondria with coupled processing of the N-terminal targeting sequence. Once imported, PNPase localized to the intermembrane space (IMS) as a peripheral membrane protein in a multimeric complex. Apoptotic stimuli caused PNPase mobilization following cytochrome c release, which supported an IMS localization and provided a potential route for interactions with cytosolic TCL1. Consistent with its IMS localization, PNPase knockdown with RNA interference did not affect mitochondrial RNA levels. However, PNPase reduction impaired mitochondrial electrochemical membrane potential, decreased respiratory chain activity, and was correlated with altered mitochondrial morphology. This resulted in F o F 1 -ATP synthase instability, impaired ATP generation, lactate accumulation, and AMP kinase phosphorylation with reduced cell proliferation. Combined, the data demonstrate an unexpected IMS localization and a key role for PNPase in maintaining mitochondrial homeostasis.