TORC1 signaling modulates Cdk8-dependent GAL gene expression in Saccharomyces cerevisiae

Cdk8 of the RNA Polymerase II mediator kinase complex regulates gene expression by phosphorylating sequence-specific transcription factors. This function is conserved amongst eukaryotes, but the signals and mechanisms regulating Cdk8 activity and phosphorylation of its substrates are unknown. Full induction of the GAL genes in yeast requires phosphorylation of the transcriptional activator Gal4 by Cdk8. We used a screen to identify regulators of the Cdk8-dependent phosphorylation on Gal4, from which we identified multiple mutants with defects in TORC1 signaling. One mutant, designated gal four throttle 1 (gft1) was identified as a recessive allele of hom3, encoding aspartokinase, and mutations in hom3 caused effects typical of inhibition of TORC1, including rapamycin sensitivity and enhanced nuclear localization of the TORC1-responsive transcription factor Gat1. Mutations in hom3 also inhibit phosphorylation of Gal4 in vivo at the Cdk8-dependent site on Gal4, as did mutations of tor1, but these mutations did not affect activity of Cdk8 assayed in vitro. Disruption of cdc55, encoding a regulatory subunit of the TORC1-regulated protein phosphatase PP2A, suppressed the effect of hom3 and tor1 mutations on GAL expression, and also restored phosphorylation of Gal4 at the Cdk8-dependent site in vivo. These observations demonstrate that TORC1 signaling regulates GAL induction through the activity of PP2A/Cdc55, and suggest that Cdk8-dependent phosphorylation of Gal4 is opposed by PP2A/Cdc55 dephosphorylation. These results provide insight into how induction of transcription by a specific inducer can be modulated by global nutritional signals through regulation of Cdk8-dependent phosphorylation.

NAD(H) Regulates the Permeability Transition Pore in Mitochondria through an External Site

The opening of the permeability transition pore (mPTP) in mitochondria initiates cell death in numerous diseases. The regulation of mPTP by NAD(H) in the mitochondrial matrix is well established; however, the role of extramitochondrial (cytosolic) NAD(H) is still unclear. We studied the effect of added NADH and NAD+ on: (1) the Ca2+-retention capacity (CRC) of isolated rat liver, heart, and brain mitochondria; (2) the Ca2+-dependent mitochondrial swelling in media whose particles can (KCl) or cannot (sucrose) be extruded from the matrix by mitochondrial carriers; (3) the Ca2+-dependent mitochondrial depolarization and the release of entrapped calcein from mitochondria of permeabilized hepatocytes; and (4) the Ca2+-dependent mitochondrial depolarization and subsequent repolarization. NADH and NAD+ increased the CRC of liver, heart, and brain mitochondria 1.5–2.5 times, insignificantly affecting the rate of Ca2+-uptake and the free Ca2+ concentration in the medium. NAD(H) suppressed the Ca2+-dependent mitochondrial swelling both in KCl- and sucrose-based media but did not induce the contraction and repolarization of swollen mitochondria. By contrast, EGTA caused mitochondrial repolarization in both media and the contraction in KCl-based medium only. NAD(H) delayed the Ca2+-dependent depolarization and the release of calcein from individual mitochondria in hepatocytes. These data unambiguously demonstrate the existence of an external NAD(H)-dependent site of mPTP regulation.

Binding of gephyrin to microtubules is regulated by its phosphorylation at Ser270

Gephyrin is a multifunctional scaffolding protein anchoring glycine- and subtypes of GABA type A- receptors at inhibitory postsynaptic membrane specializations by binding to the microtubule (MT) and/or the actin cytoskeleton. However, the conditions under which gephyrin can bind to MTs and its regulation are currently unknown. Here, we demonstrate that during the purification of MTs from rat brain by sedimentation of polymerized tubulin using high-speed centrifugation a fraction of gephyrin was bound to MTs, whereas gephyrin phosphorylated at the CDK5-dependent site Ser270 was detached from MTs and remained in the soluble protein fraction. Moreover, after collybistin fostered phosphorylation at Ser270 the binding of a recombinant gephyrin to MTs was strongly reduced in co-sedimentation assays. Correspondingly, upon substitution of wild-type gephyrin with recombinant gephyrin carrying alanine mutations at putative CDK5 phosphorylation sites the binding of gephyrin to MTs was increased. Furthermore, the analysis of cultured HEK293T and U2OS cells by immunofluorescence-microscopy disclosed a dispersed and punctuated endogenous gephyrin immunoreactivity co-localizing with MTs which was evidently not phosphorylated at Ser270. Thus, our study provides additional evidence for the binding of gephyrin to MTs in brain tissue and in in vitro cell systems. More importantly, our findings indicate that gephyrin-MT binding is restricted to a specific gephyrin fraction and depicts phosphorylation of gephyrin as a regulatory mechanism of this process by showing that soluble gephyrin detached from MTs can be detected specifically with the mAb7a antibody, which recognizes the Ser270 phosphorylated- version of gephyrin.

Identification of new OPA1 cleavage site reveals that short isoforms regulate mitochondrial fusion

OPA1, a large GTPase of the dynamin superfamily, mediates fusion of the mitochondrial inner membranes, regulates cristae morphology, and maintains respiratory chain function. Inner-membrane-anchored long forms of OPA1 (l-OPA1) are proteolytically processed by the OMA1 or YME1L proteases, acting at cleavage sites S1 and S2 respectively, to produce short forms (s-OPA1). In both mouse and human, half of the mRNA splice forms of Opa1 are constitutively processed to yield exclusively s-OPA1. However, the function of s-OPA1 in mitochondrial fusion has been debated, because in some stress conditions, s-OPA1 is dispensable for fusion. By constructing cells in which the Opa1 locus no longer produces transcripts with S2 cleavage sites, we generated a simplified system to identify the new YME1L-dependent site S3 that mediates constitutive and complete cleavage of OPA1. We show that mitochondrial morphology is highly sensitive to the ratio of l-OPA1 to s-OPA1, indicating that s-OPA1 regulates mitochondrial fusion.

Spatial regulation of clathrin-mediated endocytosis through position-dependent site maturation

During clathrin-mediated endocytosis (CME), over 50 different proteins assemble on the plasma membrane to reshape it into a cargo-laden vesicle. It has long been assumed that cargo triggers local CME site assembly in Saccharomyces cerevisiae based on the discovery that cortical actin patches, which cluster near exocytic sites, are CME sites. Quantitative imaging data reported here lead to a radically different view of which CME steps are regulated and which steps are deterministic. We quantitatively and spatially describe progression through the CME pathway and pinpoint a cargo-sensitive regulatory transition point that governs progression from the initiation phase of CME to the internalization phase. Thus, site maturation, rather than site initiation, accounts for the previously observed polarized distribution of actin patches in this organism. While previous studies suggested that cargo ensures its own internalization by regulating either CME initiation rates or frequency of abortive events, our data instead identify maturation through a checkpoint in the pathway as the cargo-sensitive step.

Time-dependent taphonomic site loss leads to spatial averaging: implications for archaeological cultures

Archaeologists typically define cultural areas on the basis of similarities between the types of material culture present in sites. The similarity is assessed in order of discovery, with newer sites being evaluated against older ones. Despite evidence for time-dependent site loss due to taphonomy, little attention has been paid to how this impacts archaeological interpretations about the spatial extents of material culture similarity. This paper tests the hypothesis that spatially incomplete datasets result in detection of larger regions of similarity. To avoid assumptions of cultural processes, we apply subsampling algorithms to a naturally occurring, spatially distributed dataset of soil types. We show that there is a negative relationship between the percentage of points used to evaluate similarity across space and the absolute distances to the first minimum in similarity for soil classifications at multiple spatial scales. This negative relationship indicates that incomplete spatial datasets lead to an overestimation of the area over which things are similar. Moreover, the location of the point from which the calculation begins can determine the size of the region of similarity. This has important implications for how we interpret the spatial extent of similarity in material culture over large distances in prehistory.

DNAzyme-powered nucleic acid release from solid supports

Here, we demonstrate use of a Mg2+-dependent, site-specific DNA enzyme (DNAzyme) to cleave oligos from polyacrylamide gel beads, which is suitable for use in drop-based assays.