Irit Orr

VDAC/porin is present in sarcoplasmic reticulum from skeletal muscle

In this study we demonstrate the existence of a protein with properties of the voltage‐dependent anion channel (VDAC) in the sarcoplasmic reticulum (SR) using multiple approaches as summarized in the following: (a) 35 and 30 kDa proteins in different SR preparations, purified from other membranal systems by Ca2+/oxalate loading and sedimentation through 55% sucrose, cross‐react with four different VDAC monoclonal antibodies. (b) Amino acid sequences of three peptides derived from the SR 35 kDa protein are identical to the sequences present in VDAC1 isoform. (c) Similar to the mitochondrial VDAC, the SR protein is specifically labeled by [14C]DCCD. (d) Using a new method, a 35 kDa protein has been purified from SR and mitochondria with a higher yield for the SR. (e) Upon reconstitution into a planar lipid bilayer, the purified SR protein shows voltage‐dependent channel activity with properties similar to those of the purified mitochondrial VDAC or VDAC1/porin 31HL from human B lymphocytes, and its channel activity is completely inhibited by the anion transport inhibitor DIDS and about 80% by DCCD. We also demonstrate the translocation of ATP into the SR lumen and the phosphorylation of the luminal protein sarcalumenin by this ATP. Both ATP translocation and sarcalumenin phosphorylation are inhibited by DIDS, but not by atractyloside, a blocker of the ATP/ADP exchanger. These results indicate the existence of VDAC, thought to be located exclusively in mitochondria, in the SR of skeletal muscle, and its possible involvement in ATP transport. Together with recent studies on VDAC multicompartment location and its dynamic association with enzymes and channels, our findings suggest that VDAC deserves attention and consideration as a protein contributing to various cellular functions.

Intrinsic disorder in the C-terminal domain of the Shaker voltage-activated K+ channel modulates its interaction with scaffold proteins

The interaction of membrane-embedded voltage-activated potassium channels (Kv) with intracellular scaffold proteins, such as the postsynaptic density 95 (PSD-95) protein, is mediated by the channel C-terminal segment. This interaction underlies Kv channel clustering at unique membrane sites and is important for the proper assembly and functioning of the synapse. In the current study, we address the molecular mechanism underlying Kv/PSD-95 interaction. We provide experimental evidence, based on hydrodynamic and spectroscopic analyses, indicating that the isolated C-terminal segment of the archetypical Shaker Kv channel (ShB-C) is a random coil, suggesting that ShB-C belongs to the recently defined class of intrinsically disordered proteins. We show that isolated ShB-C is still able to bind its scaffold protein partner and support protein clustering in vivo, indicating that unfoldedness is compatible with ShB-C activity. Pulldown experiments involving C-terminal chains differing in flexibility or length further demonstrate that intrinsic disorder in the C-terminal segment of the Shaker channel modulates its interaction with the PSD-95 protein. Our results thus suggest that the C-terminal domain of the Shaker Kv channel behaves as an entropic chain and support a “fishing rod” molecular mechanism for Kv channel binding to scaffold proteins. The importance of intrinsically disordered protein segments to the complex processes of synapse assembly, maintenance, and function is discussed.

Direct analysis of cooperativity in multisubunit allosteric proteins

Allosteric regulation of protein function is a fundamental phenomenon of major importance in many cellular processes. Such regulation is often achieved by ligand-induced conformational changes in multimeric proteins that may give rise to cooperativity in protein function. At the heart of allosteric mechanisms offered to account for such phenomenon, involving either concerted or sequential conformational transitions, lie changes in intersubunit interactions along the ligation pathway of the protein. However, structure–function analysis of such homooligomeric proteins by means of mutagenesis, although it provides valuable indirect information regarding (allosteric) mechanisms of action, it does not define the contribution of individual subunits nor interactions thereof to cooperativity in protein function, because any point mutation introduced into homooligomeric proteins will be present in all subunits. Here, we present a general strategy for the direct analysis of cooperativity in multisubunit proteins that combines measurement of the effects on protein function of all possible combinations of mutated subunits with analysis of the hierarchy of intersubunit interactions, assessed by using high-order double-mutant cycle-coupling analysis. We show that the pattern of high-order intersubunit coupling can serve as a discriminative criterion for defining concerted versus sequential conformational transitions underlying protein function. This strategy was applied to the particular case of the voltage-activated potassium channel protein (Kv) to provide compelling evidence for a concerted all-or-none activation gate opening of the Kv channel pore domain. An direct and detailed analysis of the contribution of high-order intersubunit interactions to cooperativity in the function of an allosteric protein has not previously been presented.

The identification of the phosphorylated 150/160-kDa proteins of sarcoplasmic reticulum, their kinase and their association with the ryanodine receptor

In the present work we studied the relationship between the phosphorylated 150- and 160-kDa proteins and other SR proteins in the 150,000-170,000 range of molecular masses. on SDS-PAGE, the identification of their kinase, as well as the purification and structural interactions between these proteins and the rynodine receptor (RyR). The phosphorylated 150-kDa protein was identified as sarcalumenin based on: (a) its cross-reactivity with three different monoclonal antibodies specific for sarcalumenin. (b) its mobility in SDS-PAGE which was modified upon digestion with endoglycosidase H, (c) its elution from lentil-lectin column by alpha-methyl mannoside, (d) its resistance to trypsin, (e) its ability to bind Ca2+ and to stain blue with Stains-All. The phosphorylated 160-kDa protein was identified as the histidine-rich Ca2+ binding protein (HCP) based on: (a) its Ca(2+)-binding property and staining blue with Stains-All, (b) phosphorylation with the catalytic subunit of cAMP-dependent kinase. (c) its increased mobility in SDS-PAGE in the presence of Ca2+ (d) its heat stability and (e) its partial amino acid sequence. The endogenous kinase was identified as casein kinase II (CK II) based on the inhibition of the endogenous phosphorylation 160/150-kDa proteins by heparin, 5.6-dichlorobenzimidazole riboside, polyaspartyl peptide and hemin, and its ability to use [gamma-32P]GTP as the phosphate donor. The association of CK II with SR membranes, was demonstrated using specific polyclonal anti-CK II antibodies. The luminal location of CK II is suggested because CK II was extracted from the SR by l M NaCl only after their treatment with hypotonic medium, and CK II activity was inhibited with the charged inhibitors heparin and polyaspartyl peptide only after their incubation with the SR in the presence of NP-40. The 160- and 150-kDa proteins were purified on spermine-agarose column, and were phosphorylated by CK II. Like the endogenous phosphorylation of the 150/160-kDa proteins in SR. the phosphorylation of the purified proteins by CK II was inhibited by La3+ (Cl50 = 4 microM) and hemin. The results suggest the phosphorylation of the luminally located sarcalumenin and HCP with CK II.

Modulation of the skeletal muscle ryanodine receptor by endogenous phosphorylation of 160/150-kDa proteins of the sarcoplasmic reticulum

This paper demonstrates and characterizes the inhibition of ryanodine binding caused by the phosphorylation of the 160/150-kDa proteins in skeletal muscle sarcoplasmic reticulum (SR). Inhibition of ryanodine binding was obtained by preincubation of SR membranes with ATP + NaF . The inhibition was characterized by the following findings: (a) If ATP was replaced by AdoPP[NH]P, inhibition of ryanodine binding activity was not observed. (b) The inhibitory effect of preincubation with ATP + NaF, like the phosphorylation of 150/160-kDa proteins, was Ca2+ dependent. (c) Inhibition of ryanodine binding, as the protein phosphorylation, was not observed if NaF (> 30 mM) was replaced with okadaic acid. (d) The optimal pH for the inhibition and the phosphorylation was about 7.0. (e) Both the phosphorylation of the 160/150-kDa proteins and inhibition of ryanodine binding were prevented by dichlorobenzimidazole riboside and hemin, inhibitors of casein kinase II. (f) Dephosphorylation of the 160/150-kDa proteins prevented the inhibition of ryanodine binding. (g) The presence of NP-40 during the phosphorylation prevented both the 160/150-kDa phosphorylation and the inhibition of ryanodine binding. Furthermore, a linear relationship was obtained between the degree of ryanodine binding inhibition and the level of phosphorylation of the 160/150-kDa proteins, as controlled by ATP or NaF concentrations. The binding affinity for Ca2+ of the ryanodine receptor (RyR) was modified by phosphorylation of the 160/150-kDa proteins, decreasing by up to 100-fold. The phosphorylation of the SR membranes resulted in an elimination of ryanodine binding sites with slight effect on the ryanodine binding affinity. These results suggest the modulation of the properties of the RyR by phosphorylation/dephosphorylation of the 160/150-kDa proteins. The identification of the phosphorylated 160/150-kDa proteins, their kinase, and the structural interactions between them and the RyR are presented in the accompanying paper.

The eIF3 complex of Leishmania—subunit composition and mode of recruitment to different cap-binding complexes

Eukaryotic initiation factor 3 (eIF3) is a multi-protein complex and a key participant in the assembly of the translation initiation machinery. In mammals, eIF3 comprises 13 subunits, most of which are characterized by conserved structural domains. The trypanosomatid eIF3 subunits are poorly conserved. Here, we identify 12 subunits that comprise the Leishmania eIF3 complex (LeishIF3a-l) by combining bioinformatics with affinity purification and mass spectrometry analyses. These results highlight the strong association of LeishIF3 with LeishIF1, LeishIF2 and LeishIF5, suggesting the existence of a multi-factor complex. In trypanosomatids, the translation machinery is tightly regulated in the different life stages of these organisms as part of their adaptation and survival in changing environments. We, therefore, addressed the mechanism by which LeishIF3 is recruited to different mRNA cap-binding complexes. A direct interaction was observed in vitro between the fully assembled LeishIF3 complex and recombinant LeishIF4G3, the canonical scaffolding protein of the cap-binding complex in Leishmania promastigotes. We further highlight a novel interaction between the C-terminus of LeishIF3a and LeishIF4E1, the only cap-binding protein that efficiently binds the cap structure under heat shock conditions, anchoring a complex that is deficient of any MIF4G-based scaffolding subunit.

Entropic clocks in the service of electrical signaling: ‘Ball and chain’ mechanisms for ion channel inactivation and clustering

Electrical signaling in the nervous system relies on action potential generation, propagation and transmission. Such processes are dynamic in nature and rely on precisely timed events associated with voltage‐dependent ion channel conformational transitions between their primary open, closed and inactivated states and clustering at unique membrane sites. In voltage‐dependent potassium (Kv) channels, fast inactivation and clustering processes rely on entropic clock chains as described by ‘ball and chain’ mechanisms, suggesting important roles for such chains in electrical signaling. Here, we consider evidence supporting the proposed ‘ball and chain’ mechanisms for Kv channel fast inactivation and clustering associated with intrinsically disorderedN‐ andC‐terminal regions of the protein, respectively. Based on this comparison, we delineate the requirements that argue for such a process and establish the thermodynamic signature of a ‘ball and chain’ mechanism. Finally, we demonstrate how ‘chain’‐level alternative splicing of the Kv channel gene modulates the entropic clock‐based ‘ball and chain’ inactivation and clustering channel functions underlying changes in electrical signaling. As such, the Kv channel model system exemplifies how linkage between alternative splicing and intrinsic disorder enables functional diversity.

Alternative Splicing Modulates Kv Channel Clustering through a Molecular ‘Ball and Chain’ Mechanism

Ion channel clustering at the post-synaptic density (PSD) serves a fundamental role in action potential transmission. In voltage-activated potassium channels (Kv), this process is mediated by interaction of the C-terminal tail with scaffold proteins by a currently unclear mechanism. Here, we show that interaction between the prototypical Shaker Kv channel and the PSD-95 scaffold protein is entropy-controlled and modulated by the length of the intrinsically disordered channel tail. We further show that the Kv channel tail functions as entropic clock that times scaffold protein binding. Based on these observations, we propose a ‘ball and chain’ mechanism to explain C-terminal-based Kv channel binding to scaffold proteins, analogous to the classical N-type mechanism that describes channel fast inactivation. The physiological relevance of this mechanism is demonstrated by showing that alternative splicing in the Shaker Kv channel gene, producing channel variants with distinct C-terminal tail lengths, exhibit distinct scaffold protein-mediated channel cell surface expression and clustering patterns that correlate with differences in affinity of the variants to PSD-95. We suggest that modulating channel clustering by specific spatial-temporal variant targeting serves a fundamental role in nervous system development and tuning.