Krisztina Káldi

Transcriptional Feedback of Neurospora Circadian Clock Gene by Phosphorylation-Dependent Inactivation of Its Transcription Factor

The circadian clock protein Frequency (FRQ) feedback-regulates its own expression by inhibiting its transcriptional activator, White Collar Complex (WCC). We present evidence that FRQ regulates the bulk of WCC through modulation of its phosphorylation status rather than via direct complex formation. In the absence of FRQ, WCC is hypophosphorylated and transcriptionally active, while WCC is hyperphosphorylated and transcriptionally inactive when FRQ is expressed. The phosphorylation status of WCC changes rhythmically over a circadian cycle. Dephosphorylation and activation of WCC depend on protein phosphatase 2A (PP2A), and WCC is a substrate of PP2A in vitro. Hypophosphorylated WCC binds to the clock box of the frq promoter even in the presence of FRQ, while binding of hyperphosphorylated WCC is compromised even when FRQ is depleted. We propose that negative feedback in the circadian clock of Neurospora is mediated by FRQ, which rhythmically promotes phosphorylation of WCC, functionally equivalent to a cyclin recruiting cyclin-dependent kinase to its targets.

Tim23 Links the Inner and Outer Mitochondrial Membranes

Tim23, a key component of the mitochondrial preprotein translocase, is anchored in the inner membrane by its C-terminal domain and exposes an intermediate domain in the intermembrane space that functions as a presequence receptor. We show that the N-terminal domain of Tim23 is exposed on the surface of the outer membrane. The two-membrane-spanning topology of Tim23 is a novel characteristic in membrane biology. By the simultaneous integration into two membranes, Tim23 forms contacts between the outer and inner mitochondrial membranes. Tethering the inner membrane translocase to the outer membrane facilitates the transfer of precursor proteins from the TOM complex to the TIM23 complex and increases the efficiency of protein import.

The role of the TIM8–13 complex in the import of Tim23 into mitochondria

Tim8 and Tim13 are non‐essential, conserved proteins of the mitochondrial intermembrane space, which are organized in a hetero‐oligomeric complex. They are structurally related to Tim9 and Tim10, essential components of the import machinery for mitochondrial carrier proteins. Here we show that the TIM8–13 complex interacts with translocation intermediates of Tim23, which are partially translocated across the outer membrane but not with fully imported or assembled Tim23. The TIM8–13 complex binds to the N‐terminal or intermediate domain of Tim23. It traps the incoming precursor in the intermembrane space thereby preventing retrograde translocation. The TIM8–13 complex is strictly required for import of Tim23 under conditions when a low membrane potential exists in the mitochondria. The human homologue of Tim8 is encoded by the DDP1 (deafness/dystonia peptide 1) gene, which is associated with the Mohr–Tranebjaerg syndrome (MTS), a progressive neurodegenerative disorder leading to deafness. It is demonstrated that import of human Tim23 is dependent on a high membrane potential. A mechanism to explain the pathology of MTS is discussed.

Photoadaptation in Neurospora by Competitive Interaction of Activating and Inhibitory LOV Domains

Light responses and photoadaptation of Neurospora depend on the photosensory light-oxygen-voltage (LOV) domains of the circadian transcription factor White Collar Complex (WCC) and its negative regulator VIVID (VVD). We found that light triggers LOV-mediated dimerization of the WCC. The activated WCC induces expression of VVD, which then disrupts and inactivates the WCC homodimers by the competitive formation of WCC-VVD heterodimers, leading to photoadaptation. During the day, expression levels of VVD correlate with light intensity, allowing photoadaptation over several orders of magnitude. At night, previously synthesized VVD serves as a molecular memory of the brightness of the preceding day and suppresses responses to light cues of lower intensity. We show that VVD is essential to discriminate between day and night, even in naturally ambiguous photoperiods with moonlight.

Interlocked feedback loops of the circadian clock of Neurospora crassa

Circadian clocks drive daily rhythms in physiology and behaviour, and thus allow organisms to better adapt to rhythmic changes in the environment. Circadian oscillators are cell‐autonomous systems, which generate via transcriptional, post‐transcriptional, translational and post‐translational control mechanisms a daily activity‐rhythm of a circadian transcription factor complex. According to recent models, this complex of transcription factors controls directly or indirectly expression of a large number of genes, and thus generates the potential to modulate physiological processes in a rhythmic fashion. The basic principles of the generation of circadian oscillation are similar in all eukaryotic systems. The circadian clock of the filamentous fungus Neurospora crassa is well characterized at the molecular level. Focusing on the molecular properties, interactions and post‐translational modifications of the core Neurospora clock proteins WHITE COLLAR‐1, WHITE COLLAR‐2, FREQUENCY and VIVID, this review summarizes our knowledge of the molecular basis of circadian time keeping in Neurospora. Moreover, we discuss the mechanisms by which environmental cues like light and temperature entrain and reset this circadian system.

Biogenesis of Tim23 and Tim17, integral components of the TIM machinery for matrix‐targeted preproteins

We analysed the import pathway of Tim23 and of Tim17, components of the mitochondrial import machinery for matrix‐targeted preproteins. Tim23 contains two independent import signals. One is located within the first 62 amino acid residues of the hydrophilic domain that, in the assembled protein, is exposed to the intermembrane space. This signal mediates translocation of Tim23 across the outer membrane independently of the membrane potential, ΔΨ. A second import signal is located in the C‐terminal membrane‐integrated portion of Tim23. It mediates translocation across the outer membrane and insertion into the inner membrane in a strictly ΔΨ‐dependent fashion. Structurally, Tim17 is related to Tim23 but lacks a hydrophilic domain. It contains an import signal in the C‐terminal half and its import requires ΔΨ. The ΔΨ‐dependent import signals of Tim23 and Tim17 are located at corresponding sites in these two homologous proteins. They exhibit features reminiscent of the positively charged N‐terminal presequences of matrix‐targeted precursors. Import of Tim23 and its insertion into the inner membrane requires Tim22 but not functional Tim23. Thus, biogenesis of the Tim23·17 complex depends on the Tim22 complex, which is the translocase identified as mediating the import of carrier proteins.

Transcriptional regulation of the Neurospora circadian clock gene wc-1 affects the phase of circadian output

WHITE COLLAR‐1 (WC‐1) is the limiting component of the White Collar Complex (WCC) controlling expression of the Neurospora circadian clock protein Frequency (FRQ). Accumulation of WC‐1 is supported by FRQ on a post‐transcriptional level. Here, we show that transcription of wc‐1 is organized in a complex way. Three promoters drive transcription of wc‐1. Pdist is dependent on WCC. Pprox is independent of WCC in darkness, but inducible by light in a WCC‐dependent manner. A third promoter, Pint, is located in the wc‐1 open reading frame and promotes expression of an amino‐terminally truncated WC‐1 isoform of unknown function. Expression of wc‐1 by Pdist or Pprox alone, or by a heterologous promoter, affects the entrained phase of circadian conidiation and the response of Neurospora to light. Our results indicate that transcriptional regulation of wc‐1 is required to modulate the circadian phase of clock output.

Social jetlag negatively correlates with academic performance in undergraduates

Discrepancies between sleep timing on workdays and weekends, also known as social jetlag (SJL), affect the majority of the population and have been found to be associated with increased health risk and health-impairing behaviors. In this study, we explored the relationship between SJL and academic performance in a sample of undergraduates of the Semmelweis University. We assessed SJL and other sleep-related parameters with the Munich ChronoType Questionnaire (MCTQ) (n = 753). Academic performance was measured by the average grade based on weekly test results as well as scores acquired on the final test (n = 247). The average mid-sleep point on free days in the Hungarian sample fits well the regression line plotted for longitudes within the Central European Time Zone and chronotypes, confirming that sunlight has a major impact on chronotype. Multivariate analysis showed negative effect of SJL on the weekly average grade (p = 0.028, n = 247) during the lecture term with its highly regular teaching schedules, while this association disappeared in the exam period (p = 0.871, n = 247) when students had no scheduled obligations (lower SJL). We also analyzed the relationship between the time of the weekly tests and academic performance and found that students with later sleep times on free days achieved worse in the morning (p = 0.017, n = 129), while the inverse tendency was observed for the afternoon test-takers (p = 0.10, n = 118). We did not find significant association between academic performance and sleep duration or sleep debt on work days. Our data suggest that circadian misalignment can have a significant negative effect on academic performance. One possible reason for this misalignment is socially enforced sleep times.

Sec14 Homology Domain Targets p50RhoGAP to Endosomes and Provides a Link between Rab and Rho GTPases

Sec14 protein was first identified in Saccharomyces cerevisiae, where it serves as a phosphatidylinositol transfer protein that is essential for the transport of secretory proteins from the Golgi complex. A protein domain homologous to Sec14 was identified in several mammalian proteins that regulates Rho GTPases, including exchange factors and GTPase activating proteins. P50RhoGAP, the first identified GTPase activating protein for Rho GTPases, is composed of a Sec14-like domain and a Rho-GTPase activating protein (GAP) domain. The biological function of its Sec14-like domain is still unknown. Here we show that p50RhoGAP is present on endosomal membranes, where it colocalizes with internalized transferrin receptor. We demonstrate that the Sec14-like domain of P50RhoGAP is responsible for the endosomal targeting of the protein. We also show that overexpression of p50RhoGAP or its Sec14-like domain inhibits transferrin uptake. Furthermore, both P50RhoGAP and its Sec14-like domain show colocalization with small GTPases Rab11 and Rab5. We measured bioluminescence resonance energy transfer between p50RhoGAP and Rab11, indicating that these proteins form molecular complex in vivo on endosomal membranes. The interaction was mediated by the Sec 14-like domain of p50RhoGAP. Our results indicate that Sec14-like domain, which was previously considered as a phospholipid binding module, may have a role in the mediation of protein-protein interactions. We suggest that p50RhoGAP provides a link between Rab and Rho GTPases in the regulation of receptor-mediated endocytosis.

Membrane topology of the 22 kDa integral peroxisomal membrane protein

In order to study the membrane topology and the possible function of the rat liver 22 kDa integral peroxisomal membrane protein (PMP 22) at a molecular level, we have cloned PMP 22 from a λgt11 expression library and sequenced its cDNA. Hydropathy analysis of the deduced primary structure indicates 4 putative transmembrane segments. The accessibility to exogenous aminopeptidase of PMP 22 in intact peroxisomes suggests that the N‐terminus faces the cytosol. A model of the topology of PMP 22 in the peroxisomal membrane is discussed. Homology studies revealed a striking similarity with the Mpv 17 gene product. Lack of this membrane protein causes nephrotic syndrome in mice.