The N-1-Naphthylphthalamic Acid-Binding Protein Is an Integral Membrane Protein

N-1-Naphthylphthalmic acid (NPA)-binding protein is a plasmalemma (PM) protein involved in the control of cellular auxin efflux. We re-evaluated the spatial relationship of this protein with the PM of zucchini (Cucurbita pepo L.) hypocotyls. First, Triton X-114 partitioning indicated that the NPA-binding protein was more hydrophobic than most PM proteins. Second, the NPA-binding activity was found to be resistant to proteolytic digestion in membranes. Maximum concentrations of binding sites for NPA were virtually identical in untreated and proteinase K-treated PMs: 19.2 and 20.6 pmol [3H]NPA bound/mg protein, respectively. The insensitivity of the NPA-binding protein was not due to its presence inside tightly sealed vesicles or due to lack of protease activity in the conditions tested. This protein could be made sensitive to proteolytic degradation upon solubilization by 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate in the presence of sodium molybdate. Proteinase K treatment decreased the concentration of binding sites to 0.84 pmol [3H]NPA bound/mg protein from 9.2 for untreated, solubilized PM. Third, this activity could not be solubilized by chaotropic agents or sodium carbonate treatment of intact PM. This study indicates that the NPA-binding protein may be an integral membrane protein and contradicts previously reported findings that suggested that this protein was peripheral to the PM.     

Characterization of a Light-Controlled Anion Channel in the Plasma Membrane of Mesophyll Cells of Pea

In leaf mesophyll cells of pea (Pisum sativum) light induces a transient depolarization that is at least partly due to an increased plasma membrane conductance for anions. Several channel types were identified in the plasma membrane of protoplasts from mesophyll cells using the patch-clamp technique. One of these was an anion channel with a single-channel conductance of 32 picasiemens in symmetrical 100/100 KCl solutions. In asymmetrical solutions the reversal potential indicates a high selectivity for Cl- over K+ at high cytoplasmic Cl-. At negative membrane voltages the channel openings were interrupted by very short closures. In the open channel conductance several substrates were identified. At a cytoplasmic negative logarithm of Ca concentration higher than 6.3, no channel openings were observed. When the protoplast was illuminated in the cell-attached configuration, at least one channel type had a higher opening probability. This channel can tentatively be identified as the above-described anion channel based on conductance and the characteristic short closures at negative membrane potentials. This light activation of the 32-picasiemen anion channel is a strong indication that this channel conducts the light-induced depolarizing current. Because channel activity is strongly Ca2+-dependent, a role of cytoplasmic Ca2+ concentration changes in the light activation of the conductance is discussed.     

Tim23, a Protein Import Component of the Mitochondrial Inner Membrane, Is Required for Normal Activity of the Multiple Conductance Channel, MCC

We previously showed that the conductance of a mitochondrial inner membrane channel, called MCC, was specifically blocked by peptides corresponding to mitochondrial import signals. To determine if MCC plays a role in protein import, we examined the relationship between MCC and Tim23p, a component of the protein import complex of the mitochondrial inner membrane. We find that antibodies against Tim23p, previously shown to inhibit mitochondrial protein import, inhibit MCC activity. We also find that MCC activity is altered in mitochondria isolated from yeast carrying the tim23-1 mutation. In contrast to wild-type MCC, we find that the conductance of MCC from the tim23-1 mutant is not significantly blocked by mitochondrial presequence peptides. Tim23 antibodies and the tim23-1 mutation do not, however, alter the activity of PSC, a presequence-peptide sensitive channel in the mitochondrial outer membrane. Our results show that Tim23p is required for normal MCC activity and raise the possibility that precursors are translocated across the inner membrane through the pore of MCC.     

A Mutant Plasma Membrane Protein Is Stabilized Upon Loss of Yvh1, a Novel Ribosome Assembly Factor

Pma1-10 is a mutant plasma membrane ATPase defective at the restrictive temperature in stability at the cell surface. At 37°, Pma1-10 is ubiquitinated and internalized from the plasma membrane for degradation in the vacuole. YVH1, encoding a tyrosine phosphatase, is a mutant suppressor of pma1-10; in the absence of Yvh1, Pma1-10 remains stable at the plasma membrane, thereby permitting cells to grow. The RING finger domain of Yvh1, but not its phosphatase domain, is required for removal of mutant Pma1-10 from the plasma membrane. Yvh1 is a novel ribosome assembly factor: in yvh1Δ cells, free 60S and 80S ribosomal subunits are decreased, free 40S subunits are increased, and half-mer polysomes are accumulated. Pma1-10 is also stabilized by deletion of 60S ribosomal proteins Rpl19a and Rpl35a. We propose that changes in ribosome biogenesis caused by loss of Yvh1 or specific ribosomal proteins have effects on the plasma membrane, perhaps by producing specific translational changes.     

Studies of the Mechanism of Activation of the Volume-Regulated Anion Channel in Rat Pancreatic β-Cells

There is evidence that depolarization of the pancreatic β cell by glucose involves cell swelling and activation of the volume-regulated anion channel (VRAC). However, it is unclear whether cell swelling per se or accompanying changes in intracellular osmolality and/or ionic strength are responsible for VRAC activation. VRAC activity was measured in rat β cells by conventional or perforated patch whole-cell recording. Cell volume was measured by video imaging. In conventional whole-cell recordings, VRAC activation was achieved by exposure of the cells to a hyposmotic bath solution, by application of positive pressure to the pipette, or by use of a hyperosmotic pipette solution. Increased concentrations of intracellular CsCl also caused channel activation, but with delayed kinetics. In perforated patch recordings, VRAC activation was induced by isosmotic addition of the permeable osmolytes urea, 3-Ο-methyl glucose, arginine, and NH₄Cl. These effects were all accompanied by β-cell swelling. It is concluded that increased cell volume, whether accompanied by raised intracellular osmolality or ionic strength, is a major determinant of VRAC activation in the β cell. However, increased intracellular ionic strength markedly reduced the rate of VRAC activation. These findings are consistent with the hypothesis that the accumulation of glucose metabolites in the β cell, and the resultant increase in cell volume, provides a signal coupling glucose metabolism with VRAC activation.     

ATP-Dependent Regulation of an Anion Channel at the Plasma Membrane of Protoplasts from Epidermal Cells of Arabidopsis Hypocotyls

Although Arabidopsis is the object of many genetic and molecular biology investigations, relatively few studies deal with regulation of its transmembrane ion exchanges. To clarify the role of ion transport in plant development, organ-and tissue-specific ion channels must be studied. We identified a voltage-dependent anion channel in epidermal cells of Arabidopsis hypocotyls, thus providing a new example of the occurrence of voltage-dependent anion channels in a specific plant cell type distinct from the stomatal guard cell. The Arabidopsis hypocotyl anion channel is able to function under two modes characterized by different voltage dependences and different kinetic behaviors. This switch between a fast and a slow mode is controlled by ATP. In the presence of intracellular ATP (fast mode), the channels are closed at resting potentials, and whole-cell currents activate upon depolarization. After activation, the anion current deactivates rapidly and more and more completely at potentials negative to the peak. In the absence of ATP, the current switches from this fast mode to a mode characterized by a slow and incomplete deactivation at resting potentials. In addition, the whole-cell currents can be correlated with the activity of single channels. In the outside-out configuration, the presence of ATP modulates the mean lifetimes of the open and closed states of the channel at hyperpolarized potentials, thus controlling its open probability. The fact that ATP-dependent voltage regulation was observed in both whole-cell and outside-out configurations suggests that a single type of anion channel can switch between two modes with distinct functional properties.     

The Microtubule-Binding Protein Ensconsin Is an Essential Cofactor of Kinesin-1

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Highlights? Ensconsin is required for cargo transport driven by kinesin-1 ? The C terminus of ensconsin activates kinesin-1; its N terminus binds microtubules ? Kinesin-1 mutants without autoinhibitory activity do not require ensconsin     

Mpe1, a Zinc Knuckle Protein, Is an Essential Component of Yeast Cleavage and Polyadenylation Factor Required for the Cleavage and Polyadenylation of mRNA

In Saccharomyces cerevisiae, in vitro mRNA cleavage and polyadenylation require the poly(A) binding protein, Pab1p, and two multiprotein complexes: CFI (cleavage factor I) and CPF (cleavage and polyadenylation factor). We characterized a novel essential gene, MPE1 (YKL059c), which interacts genetically with the PCF11 gene encoding a subunit of CFI. Mpe1p is an evolutionarily conserved protein, a homolog of which is encoded by the human genome. The protein sequence contains a putative RNA-binding zinc knuckle motif. MPE1 is implicated in the choice of ACT1 mRNA polyadenylation site in vivo. Extracts from a conditional mutant, mpe1-1, or from a wild-type extract immunoneutralized for Mpe1p are defective in 3'-end processing. We used the tandem affinity purification (TAP) method on strains TAP-tagged for Mpe1p or Pfs2p to show that Mpe1p, like Pfs2p, is an integral subunit of CPF. Nevertheless a stable CPF, devoid of Mpe1p, was purified from the mpe1-1 mutant strain, showing that Mpe1p is not directly involved in the stability of this complex. Consistently, Mpe1p is also not necessary for the processive polyadenylation, nonspecific for the genuine pre-mRNA 3' end, displayed by the CPF alone. However, a reconstituted assay with purified CFI, CPF, and the recombinant Pab1p showed that Mpe1p is strictly required for the specific cleavage and polyadenylation of pre-mRNA. These results show that Mpe1p plays a crucial role in 3' end formation probably by promoting the specific link between the CFI/CPF complex and pre-mRNA.     

Phospholipid Association Is Essential for Dynamin-related Protein Mgm1 to Function in Mitochondrial Membrane Fusion

Mgm1, the yeast ortholog of mammalian OPA1, is a key component in mitochondrial membrane fusion and is required for maintaining mitochondrial dynamics and morphology. We showed recently that the purified short isoform of Mgm1 (s-Mgm1) possesses GTPase activity, self-assembles into low order oligomers, and interacts specifically with negatively charged phospholipids (Meglei, G., and McQuibban, G. A. (2009) Biochemistry 48, 1774–1784). Here, we demonstrate that s-Mgm1 binds to a mixture of phospholipids characteristic of the mitochondrial inner membrane. Binding to physiologically representative lipids results in ∼50-fold stimulation of s-Mgm1 GTPase activity. s-Mgm1 point mutants that are defective in oligomerization and lipid binding do not exhibit such stimulation and do not function in vivo. Electron microscopy and lipid turbidity assays demonstrate that s-Mgm1 promotes liposome interaction. Furthermore, s-Mgm1 assembles onto liposomes as oligomeric rings with 3-fold symmetry. The projection map of negatively stained s-Mgm1 shows six monomers, consistent with two stacked trimers. Taken together, our data identify a lipid-binding domain in Mgm1, and the structural analysis suggests a model of how Mgm1 promotes the fusion of opposing mitochondrial inner membranes.Mitochondrial dynamics have been implicated in neurodegenerative diseases such as dominant optic atrophy and Parkinson disease (1, 2). Mitochondrial morphology is regulated by balanced membrane fusion and fission reactions that are orchestrated by members of the highly conserved dynamin-related protein family (3). Dynamin-related proteins are large GTPases that can self-assemble and promote membrane remodeling (4, 5). We have shown previously that the dynamin-related protein Mgm1 has GTPase activity, self-assembles into low order oligomers, and binds to negatively charged phospholipids (6). Mgm1 exists as two isoforms in the mitochondria; l-Mgm1² is anchored to the IM via a transmembrane domain, and s-Mgm1 is peripherally associated with the IM and also found in the intermembrane space. s-Mgm1 results from the regulated cleavage by the mitochondrial rhomboid protease (7, 8). It was shown recently that both isoforms are essential but have distinct roles in mitochondrial membrane fusion whereby only s-Mgm1 requires its GTPase activity (9). It is proposed that l-Mgm1 serves as a receptor for s-Mgm1 to mediate fusion of opposing membranes upon GTP hydrolysis. Here, we provide molecular data indicating that lipid binding of s-Mgm1 is required for proper membrane fusion. Furthermore, structural analysis of s-Mgm1 assembled onto liposomes suggests a model whereby stacked trimers of s-Mgm1 on opposing membranes would facilitate fusion.     

Characterization of an Anion Transporter in the Plasma Membrane of Barley Roots

To examine the relationship between H⁺-ATPase and the transportof anions, we investigated the effects of various inhibitorson the activity of the H⁺-ATPase, the transport of protons,and the transport of Cl^- ions using plasma membrane vesiclesprepared from barley roots. Some inhibitors, namely, 4,4-diisothiocyano-2,2-stilbenedisulfonate (DIDS) and Zn²⁺ ions markedly inhibited H⁺- ATPaseactivity. Other compounds, such as phenylglyoxal (PGO) and niflumicacid (NIF), inhibited H⁺-ATPase activity by 20-30%, while anthracene-9-carboxylate(A-9-C) and tetraethylammonium chloride (TEA-Cl) had littleeffect on this activity. The ATP-dependent acidification ofthe interior of vesicles was strongly dependent on the presenceof permeant anions, such as chloride (Cl^-) and nitrate (NO₃^-),and it was completely inhibited by 0.2 mM NIF. Other compounds,namely, A-9-C of 0.1 mM and TEA-Cl of 10 mM, did not affectH⁺-transport activity. The inhibition of H⁺-transport activityby NIF was observed even when the activity was assayed in thepresence of KCl, KNO₃, or bis-tris-propane (BTP)-Cl. Using ³⁶cl,we quantified Cl^--transport activity by measuring the uptakeof Cl^- ions into the plasma membrane vesicles. The uptake dependedon the potential difference across the membrane that was generatedby H⁺-ATPase; it was enhanced by an inside-positive potentialgradient. At 0.1 mM, NIF completely blocked the voltage-dependentCl^--transport activity. From these properties of the Cl^- transporterand the inhibition of H⁺-transport activity by NIF, we suggestthat H⁺-transport activity across the plasma membrane mightbe modulated by the transport of anions via a NIF-sensitiveanion-permeable transporter that acts to collapse the inside-positivepotential generated by H⁺-ATPase. (Received September 7, 1995; Accepted July 23, 1996)     

Translocation of Sphingosine Kinase 1 to the Plasma Membrane Is Mediated by Calcium- and Integrin-binding Protein 1

SK1 (sphingosine kinase 1) plays an important role in many aspects of cellular regulation. Most notably, elevated cellular SK1 activity leads to increased cell proliferation, protection from apoptosis, and induction of neoplastic transformation. We have previously shown that translocation of SK1 from the cytoplasm to the plasma membrane is integral for oncogenesis mediated by this enzyme. The molecular mechanism mediating this translocation of SK1 has remained undefined. Here, we demonstrate a direct role for CIB1 (calcium and integrin-binding protein 1) in this process. We show that CIB1 interacts with SK1 in a Ca²⁺-dependent manner at the previously identified “calmodulin-binding site” of SK1. We also demonstrate that CIB1 functions as a Ca²⁺-myristoyl switch, providing a mechanism whereby it translocates SK1 to the plasma membrane. Both small interfering RNA knockdown of CIB1 and the use of a dominant-negative CIB1 we have generated prevent the agonist-dependent translocation of SK1. Furthermore, we demonstrate the requirement of CIB1-mediated translocation of SK1 in controlling cellular sphingosine 1-phosphate generation and associated anti-apoptotic signaling.     

Hcn1 Is a Tremorgenic Genetic Component in a Rat Model of Essential Tremor

Genetic factors are thought to play a major role in the etiology of essential tremor (ET); however, few genetic changes that induce ET have been identified to date. In the present study, to find genes responsible for the development of ET, we employed a rat model system consisting of a tremulous mutant strain, TRM/Kyo (TRM), and its substrain TRMR/Kyo (TRMR). The TRM rat is homozygous for the tremor (tm) mutation and shows spontaneous tremors resembling human ET. The TRMR rat also carries a homozygous tm mutation but shows no tremor, leading us to hypothesize that TRM rats carry one or more genes implicated in the development of ET in addition to the tm mutation. We used a positional cloning approach and found a missense mutation (c. 1061 C>T, p. A354V) in the hyperpolarization-activated cyclic nucleotide-gated 1 channel (Hcn1) gene. The A354V HCN1 failed to conduct hyperpolarization-activated currents in vitro, implicating it as a loss-of-function mutation. Blocking HCN1 channels with ZD7288 in vivo evoked kinetic tremors in nontremulous TRMR rats. We also found neuronal activation of the inferior olive (IO) in both ZD7288-treated TRMR and non-treated TRM rats and a reduced incidence of tremor in the IO-lesioned TRM rats, suggesting a critical role of the IO in tremorgenesis. A rat strain carrying the A354V mutation alone on a genetic background identical to that of the TRM rats showed no tremor. Together, these data indicate that body tremors emerge when the two mutant loci, tm and Hcn1^A354V, are combined in a rat model of ET. In this model, HCN1 channels play an important role in the tremorgenesis of ET. We propose that oligogenic, most probably digenic, inheritance is responsible for the genetic heterogeneity of ET.     

STIM1 Is a Novel Component of ER-Chlamydia trachomatis Inclusion Membrane Contact Sites

Productive developmental cycle of the obligate intracellular bacterial pathogen Chlamydia trachomatis depends on the interaction of the replicative vacuole, named the inclusion, with cellular organelles. We have recently reported the formation of ER-Inclusion membrane contact sites (MCSs), where the endoplasmic reticulum (ER) is in apposition to the inclusion membrane. These platforms contain the C. trachomatis inclusion membrane protein IncD, the mammalian ceramide transfer protein CERT and the ER resident proteins VAPA/B and were proposed to play a role in the non-vesicular trafficking of lipids to the inclusion. Here, we identify STIM1 as a novel component of ER-Inclusion MCSs. STIM1, an ER calcium (Ca²⁺) sensor that relocate to ER-Plasma Membrane (PM) MCSs upon Ca²⁺ store depletion, associated with C. trachomatis inclusion. STIM1, but not the general ER markers Rtn3C and Sec61ß, was enriched at the inclusion membrane. Ultra-structural studies demonstrated that STIM1 localized to ER-Inclusion MCSs. Time-course experiments showed that STIM1, CERT and VAPB co-localized throughout the developmental cycle. By contrast, Orai1, the PM Ca²⁺ channel that interacts with STIM1 at ER-PM MCSs, did not associate with C. trachomatis inclusion. Upon ER Ca²⁺ store depletion, a pool of STIM1 relocated to ER-PM MCSs, while the existing ER-Inclusion MCSs remained enriched in STIM1. Finally, we have identified the CAD domain, which mediates STIM1-Orai1 interaction, as the minimal domain required for STIM1 enrichment at ER-Inclusion MCSs. Altogether this study identifies STIM1 as a novel component of ER-C. trachomatis inclusion MCSs. We discuss the potential role(s) of STIM1 during the infection process.     

Stu2p: A Microtubule-Binding Protein that Is an Essential Component of the Yeast Spindle Pole Body

Previously we isolated tub2-423, a cold-sensitive allele of the Saccharomyces cerevisiae gene encoding β-tubulin that confers a defect in mitotic spindle function. In an attempt to identify additional proteins that are important for spindle function, we screened for suppressors of the cold sensitivity of tub2-423 and obtained two alleles of a novel gene, STU2. STU2 is an essential gene and encodes a protein whose sequence is similar to proteins identified in a variety of organisms. Stu2p localizes primarily to the spindle pole body (SPB) and to a lesser extent along spindle microtubules. Localization to the SPB is not dependent on the presence of microtubules, indicating that Stu2p is an integral component of the SPB. Stu2p also binds microtubules in vitro. We have localized the microtubule-binding domain of Stu2p to a highly basic 100-amino acid region. This region contains two imperfect repeats; both repeats appear to contribute to microtubule binding to similar extents. These results suggest that Stu2p may play a role in the attachment, organization, and/or dynamics of microtubule ends at the SPB.     

TORC2 Plasma Membrane Localization Is Essential for Cell Viability and Restricted to a Distinct Domain

The conserved target of rapamycin (TOR) kinases regulate many aspects of cellular physiology. They exist in two distinct complexes, termed TOR complex 1 (TORC1) and TOR complex 2 (TORC2), that posses both overlapping and distinct components. TORC1 and TORC2 respond differently to the drug rapamycin and have different cellular functions: whereas the rapamycin-sensitive TORC1 controls many aspects of cell growth and has been characterized in great detail, the TOR complex 2 is less understood and regulates actin polymerization, cell polarity, and ceramide metabolism. How signaling specificity and discrimination between different input signals for the two kinase complexes is achieved is not understood. Here, we show that TORC1 and TORC2 have different localizations in Saccharomyces cerevisiae. TORC1 is localized exclusively to the vacuolar membrane, whereas TORC2 is localized dynamically in a previously unrecognized plasma membrane domain, which we term membrane compartment containing TORC2 (MCT). We find that plasma membrane localization of TORC2 is essential for viability and mediated by lipid binding of the C-terminal domain of the Avo1 subunit. From these data, we suggest that the TOR complexes are spatially separated to determine downstream signaling specificity and their responsiveness to different inputs.     

PFP1, a Gene Encoding an Epc-N Domain-Containing Protein,Is Essential for Pathogenicity of the Barley Pathogen Rhynchosporium commune

Scald caused by Rhynchosporium commune is an important foliar disease of barley. Insertion mutagenesis of R. commune generated a nonpathogenic fungal mutant which carries the inserted plasmid in the upstream region of a gene named PFP1. The characteristic feature of the gene product is an Epc-N domain. This motif is also found in homologous proteins shown to be components of histone acetyltransferase (HAT) complexes of fungi and animals. Therefore, PFP1 is suggested to be the subunit of a HAT complex in R. commune with an essential role in the epigenetic control of fungal pathogenicity. Targeted PFP1 disruption also yielded nonpathogenic mutants which showed wild-type-like growth ex planta, except for the occurrence of hyphal swellings. Complementation of the deletion mutants with the wild-type gene reestablished pathogenicity and suppressed the hyphal swellings. However, despite wild-type-level PFP1 expression, the complementation mutants did not reach wild-type-level virulence. This indicates that the function of the protein complex and, thus, fungal virulence are influenced by a position-affected long-range control of PFP1 expression.     

Polycomb Group Protein YY1 Is an Essential Regulator of Hematopoietic Stem Cell Quiescence

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KDEL-Containing Auxin-Binding Protein Is Secreted to the Plasma Membrane and Cell Wall

The auxin-binding protein ABP1 has been postulated to mediate auxin-induced cellular changes associated with cell expansion. This protein contains the endoplasmic reticulum (ER) retention signal, the tetrapeptide lysine-aspartic acid-glutamic acid-leucine (KDEL), at its carboxy terminus, consistent with previous subcellular fractionation data that indicated an ER location for ABP1. We used electron microscopic immunocytochemistry to identify the subcellular localization of ABP1. Using maize (Zea mays) coleoptile tissue and a black Mexican sweet (BMS) maize cell line, we found that ABP1 is located in the ER as expected, but is also on or closely associated with the plasma membrane and within the cell wall. Labeling of the Golgi apparatus suggests that the transport of ABP1 to the cell wall occurs via the secretory system. Inhibition of secretion of an ABP homolog into the medium of BMS cell cultures by brefeldin A, a drug that specifically blocks secretion, is consistent with this secretion pathway. The secreted protein was recognized by an anti-KDEL peptide antibody, strongly supporting the interpretation that movement of this protein out of the ER does not involve loss of the carboxy-terminal signal. Cells starved for 2,4-dichlorophenoxyacetic acid for 72 h retained less ABP in the cell and secreted more of it into the medium. The significance of our observations is 2-fold. We have identified a KDEL-containing protein that specifically escapes the ER retention system, and we provide an explanation for the apparent discrepancy that most of the ABP is located in the ER, whereas ABP and auxin act at the plasma membrane.