Role of ATP in mitochondrial protein import

The role of nucleoside triphosphates (NTPs) in the import of porin into the mitochondrial outer membrane was investigated with two forms of the porin precursor: the in vitro synthesized biosynthetic precursor (bs- porin) and a water-soluble form of porin (ws-porin) obtained by subjecting the membrane-derived porin to an acid-base treatment (exposure to trichloroacetic acid, followed by alkali and rapid neutralization). The import of ws-porin into mitochondria did not require NTPs, whereas the import of bs-porin required NTPs. In other characteristics, such as binding to a specific receptor protein on the mitochondrial surface, two-step insertion into the outer membrane, and formation of specific membrane channels, ws-porin was indistinguishable from bs-porin. Thus, the acid-base treatment applied in the preparation of ws-porin can substitute for the NTP-requiring step in mitochondrial protein import. We conclude that NTPs are required for unfolding mitochondrial precursor proteins ("translocation competent folding")

Mitochondrial porin of Neurospora crassa

cDNA encoding porin of Neurospora crassa, the major protein component of the outer mitochondrial membrane, was isolated and the nucleotide sequence was determined. The deduced protein sequence consists of 283 amino acids (29,979 daltons) and shows sequence homology of around 43% to yeast porin; however, no significant homology to bacterial porins was apparent. According to secondary structure predictions, mitochondrial porin consists mainly of membrane-spanning sided beta-sheets. Porin was efficiently synthesized in vitro from the cDNA; this allowed us to study in detail its import into mitochondria. Thereby, three characteristics of import were defined: (i) import depended on the presence of nucleoside triphosphates; (ii) involvement of a proteinaceous receptor-like component on the surface of the mitochondria was demonstrated; (iii) insertion into the outer membrane was resolved into at least two distinct steps: specific binding to high-affinity sites and subsequent assembly to the mature form

Impaired Fear Extinction Due to a Deficit in Ca2+ Influx Through L-Type Voltage-Gated Ca2+ Channels in Mice Deficient for Tenascin-C

Mice deficient in the extracellular matrix glycoprotein tenascin-C (TNC−/−) express a deficit in specific forms of hippocampal synaptic plasticity, which involve the L-type voltage-gated Ca2+ channels (L-VGCCs). The mechanisms underlying this deficit and its functional implications for learning and memory have not been investigated. In line with previous findings, we report on impairment in theta-burst stimulation (TBS)-induced long-term potentiation (LTP) in TNC−/− mice in the CA1 hippocampal region and its rescue by the L-VGCC activator Bay K-8644. We further found that the overall pattern of L-VGCC expression in the hippocampus in TNC−/− mice was normal, but Western blot analysis results uncovered upregulated expression of the Cav1.2 and Cav1.3 α-subunits of L-VGCCs. However, these L-VGCCs were not fully functional in TNC−/− mice, as demonstrated by Ca2+ imaging, which revealed a reduction of nifedipine-sensitive Ca2+ transients in CA1 pyramidal neurons. TNC−/− mice showed normal learning and memory in the contextual fear conditioning paradigm but impaired extinction of conditioned fear responses. Systemic injection of the L-VGCC blockers nifedipine and diltiazem into wild-type mice mimicked the impairment of fear extinction observed in TNC−/− mice. The deficiency in TNC−/− mice substantially occluded the effects of these drugs. Our results suggest that TNC-mediated modulation of L-VGCC activity is essential for fear extinction

The KDET Motif in the Intracellular Domain of the Cell Adhesion Molecule L1 Interacts with Several Nuclear, Cytoplasmic, and Mitochondrial Proteins Essential for Neuronal Functions

Abnormal functions of the cell adhesion molecule L1 are linked to several neural diseases. Proteolytic L1 fragments were reported to interact with nuclear and mitochondrial proteins to regulate events in the developing and the adult nervous system. Recently, we identified a 55 kDa L1 fragment (L1-55) that interacts with methyl CpG binding protein 2 (MeCP2) and heterochromatin protein 1 (HP1) via the KDET motif. We now show that L1-55 also interacts with histone H1.4 (HistH1e) via this motif. Moreover, we show that this motif binds to NADH dehydrogenase ubiquinone flavoprotein 2 (NDUFV2), splicing factor proline/glutamine-rich (SFPQ), the non-POU domain containing octamer-binding protein (NonO), paraspeckle component 1 (PSPC1), WD-repeat protein 5 (WDR5), heat shock cognate protein 71 kDa (Hsc70), and synaptotagmin 1 (SYT1). Furthermore, applications of HistH1e, NDUFV2, SFPQ, NonO, PSPC1, WDR5, Hsc70, or SYT1 siRNAs or a cell-penetrating KDET-carrying peptide decrease L1-dependent neurite outgrowth and the survival of cultured neurons. These findings indicate that L1’s KDET motif binds to an unexpectedly large number of molecules that are essential for nervous system-related functions, such as neurite outgrowth and neuronal survival. In summary, L1 interacts with cytoplasmic, nuclear and mitochondrial proteins to regulate development and, in adults, the formation, maintenance, and flexibility of neural functions

The Interactions of the 70 kDa Fragment of Cell Adhesion Molecule L1 with Topoisomerase 1, Peroxisome Proliferator-Activated Receptor γ and NADH Dehydrogenase (Ubiquinone) Flavoprotein 2 Are Involved in Gene Expression and Neuronal L1-Dependent Functions

The cell adhesion molecule L1 is essential not only for neural development, but also for synaptic functions and regeneration after trauma in adulthood. Abnormalities in L1 functions cause developmental and degenerative disorders. L1’s functions critically depend on proteolysis which underlies dynamic cell interactions and signal transduction. We showed that a 70 kDa fragment (L1-70) supports mitochondrial functions and gene transcription. To gain further insights into L1-70’s functions, we investigated several binding partners. Here we show that L1-70 interacts with topoisomerase 1 (TOP1), peroxisome proliferator-activated receptor γ (PPARγ) and NADH dehydrogenase (ubiquinone) flavoprotein 2 (NDUFV2). TOP1, PPARγ and NDUFV2 siRNAs reduced L1-dependent neurite outgrowth, and the topoisomerase inhibitors topotecan and irinotecan inhibited L1-dependent neurite outgrowth, neuronal survival and migration. In cultured neurons, L1 siRNA reduces the expression levels of the long autism genes neurexin-1 (Nrxn1) and neuroligin-1 (Nlgn1) and of the mitochondrially encoded gene NADH:ubiquinone oxidoreductase core subunit 2 (ND2). In mutant mice lacking L1-70, Nrxn1 and Nlgn1, but not ND2, mRNA levels are reduced. Since L1-70’s interactions with TOP1, PPARγ and NDUFV2 contribute to the expression of two essential long autism genes and regulate important neuronal functions, we propose that L1 may not only ameliorate neurological problems, but also psychiatric dysfunctions

Interactions between the Polysialylated Neural Cell Adhesion Molecule and the Transient Receptor Potential Canonical Channels 1, 4, and 5 Induce Entry of Ca2+ into Neurons

The neural cell adhesion molecule (NCAM) plays important functional roles in the developing and mature nervous systems. Here, we show that the transient receptor potential canonical (TRPC) ion channels TRPC1, −4, and −5 not only interact with the intracellular domains of the transmembrane isoforms NCAM140 and NCAM180, but also with the glycan polysialic acid (PSA) covalently attached to the NCAM protein backbone. NCAM antibody treatment leads to the opening of TRPC1, −4, and −5 hetero- or homomers at the plasma membrane and to the influx of Ca2+ into cultured cortical neurons and CHO cells expressing NCAM, PSA, and TRPC1 and −4 or TRPC1 and −5. NCAM-stimulated Ca2+ entry was blocked by the TRPC inhibitor Pico145 or the bacterial PSA homolog colominic acid. NCAM-stimulated Ca2+ influx was detectable neither in NCAM-deficient cortical neurons nor in TRPC1/4- or TRPC1/5-expressing CHO cells that express NCAM, but not PSA. NCAM-induced neurite outgrowth was reduced by TRPC inhibitors and a function-blocking TRPC1 antibody. A characteristic signaling feature was that extracellular signal-regulated kinase 1/2 phosphorylation was also reduced by TRPC inhibitors. Our findings indicate that the interaction of NCAM with TRPC1, −4, and −5 contributes to the NCAM-stimulated and PSA-dependent Ca2+ entry into neurons thereby influencing essential neural functions