Ca2+/Calmodulin Binding to STIM1 Hydrophobic Residues Facilitates Slow Ca2+-Dependent Inactivation of the Orai1 Channel.

BACKGROUND/AIMS: Store-operated Ca2+ entry (SOCE) through plasma membrane Ca2+ channel Orai1 is essential for many cellular processes. SOCE, activated by ER Ca2+ store-depletion, relies on the gating function of STIM1 Orai1-activating region SOAR of the ER-anchored Ca2+-sensing protein STIM1. Electrophysiologically, SOCE is characterized as Ca2+ release-activated Ca2+ current (ICRAC). A major regulatory mechanism that prevents deleterious Ca2+ overload is the slow Ca2+-dependent inactivation (SCDI) of ICRAC. Several studies have suggested a role of Ca2+/calmodulin (Ca2+/CaM) in triggering SCDI. However, a direct contribution of STIM1 in regulating Ca2+/CaM-mediated SCDI of ICRAC is as yet unclear. METHODS: The Ca2+/CaM binding to STIM1 was tested by pulling down recombinant GFP-tagged human STIM1 C-terminal fragments on CaM sepharose beads. STIM1 was knocked out by CRISPR/Cas9 technique in HEK293 cells stably overexpressing human Orai1. Store-operated Ca2+ influx was measured using Fluorometric Imaging Plate Reader and whole-cell patch clamp in cells transfected with STIM1 CaM binding mutants. The involvement of Ca2+/CaM in SCDI was investigated by including recombinant human CaM in patch pipette in electrophysiology. RESULTS: Here we identified residues Leu374/Val375 (H1) and Leu390/Phe391 (H2) within SOAR that serve as hydrophobic anchor sites for Ca2+/CaM binding. The bifunctional H2 site is critical for both Orai1 activation and Ca2+/CaM binding. Single residue mutations of Phe391 to less hydrophobic residues significantly diminished SOCE and ICRAC, independent of Ca2+/CaM. Hence, the role of H2 residues in Ca2+/CaM-mediated SCDI cannot be precisely evaluated. In contrast, the H1 site controls exclusively Ca2+/CaM binding and subsequently SCDI, but not Orai1 activation. V375A but not V375W substitution eliminated SCDI of ICRAC caused by Ca2+/CaM, proving a direct role of STIM1 in coordinating SCDI. CONCLUSION: Taken together, we propose a mechanistic model, wherein binding of Ca2+/CaM to STIM1 hydrophobic anchor residues, H1 and H2, triggers SCDI by disrupting the functional interaction between STIM1 and Orai1. Our findings reveal how STIM1, Orai1, and Ca2+/CaM are functionally coordinated to control ICRAC.

The SESA network links duplication of the yeast centrosome with the protein translation machinery.

The yeast spindle pole body (SPB), the functional equivalent of mammalian centrosome, duplicates in G1/S phase of the cell cycle and then becomes inserted into the nuclear envelope. Here we describe a link between SPB duplication and targeted translation control. When insertion of the newly formed SPB into the nuclear envelope fails, the SESA network comprising the GYF domain protein Smy2, the translation inhibitor Eap1, the mRNA-binding protein Scp160 and the Asc1 protein, specifically inhibits initiation of translation of POM34 mRNA that encodes an integral membrane protein of the nuclear pore complex, while having no impact on other mRNAs. In response to SESA, POM34 mRNA accumulates in the cytoplasm and is not targeted to the ER for cotranslational translocation of the protein. Reduced level of Pom34 is sufficient to restore viability of mutants with defects in SPB duplication. We suggest that the SESA network provides a mechanism by which cells can regulate the translation of specific mRNAs. This regulation is used to coordinate competing events in the nuclear envelope.

Multiple glutathione disulfide removal pathways mediate cytosolic redox homeostasis.

Glutathione is central to cellular redox chemistry. The majority of glutathione redox research has been based on the chemical analysis of whole-cell extracts, which unavoidably destroy subcellular compartment-specific information. Compartment-specific real-time measurements based on genetically encoded fluorescent probes now suggest that the cytosolic glutathione redox potential is about 100 mV more reducing than previously thought. Using these probes in yeast, we show that even during severe oxidative stress, the cytosolic glutathione disulfide (GSSG) concentration is much more tightly regulated than expected and provides a mechanistic explanation for the discrepancy with conventional measurements. GSSG that is not immediately reduced in the cytosol is rapidly transported into the vacuole by the ABC-C transporter Ycf1. The amount of whole-cell GSSG is entirely dependent on Ycf1 and uninformative about the cytosolic glutathione pool. Applying these insights, we identify Trx2 and Grx2 as efficient backup systems to glutathione reductase for cytosolic GSSG reduction.

Di-arginine signals and the K-rich domain retain the Ca²âº sensor STIM1 in the endoplasmic reticulum.

STIM1 is a core component of the store-operated Ca²âº-entry channel involved in Ca²âº-signaling with an important role in the activation of immune cells and many other cell types. In response to cell activation, STIM1 protein senses low Ca²âº concentration in the lumen of the endoplasmic reticulum (ER) and activates the channel protein Orai1 in the plasma membrane by direct physical contact. The related protein STIM2 functions similar but its physiological role is less well defined. We found that STIM2, but not STIM1, contains a di-lysine ER-retention signal. This restricts the function of STIM2 as Ca²âº sensor to the ER while STIM1 can reach the plasma membrane. The intracellular distribution of STIM1 is regulated in a cell-cycle-dependent manner with cell surface expression of STIM1 during mitosis. Efficient retention of STIM1 in the ER during interphase depends on its lysine-rich domain and a di-arginine ER retention signal. Store-operated Ca²âº-entry enhanced ER retention, suggesting that trafficking of STIM1 is regulated and this regulation contributes to STIM1s role as multifunctional component in Ca²âº-signaling.

Binding of plasma membrane lipids recruits the yeast integral membrane protein Ist2 to the cortical ER.

Recruitment of cytosolic proteins to individual membranes is governed by a combination of protein-protein and protein-membrane interactions. Many proteins recognize phosphatidylinositol 4,5-bisphosphate [PI(4,5)P(2)] at the cytosolic surface of the plasma membrane (PM). Here, we show that a protein-lipid interaction can also serve as a dominant signal for the sorting of integral membrane proteins. Interaction with phosphatidly-inositolphosphates (PIPs) at the PM is involved in the targeting of the polytopic yeast protein Ist2 to PM-associated domains of the cortical endoplasmic reticulum (ER). Moreover, binding of PI(4,5)P(2) at the PM functions as a dominant mechanism that targets other integral membrane proteins to PM-associated domains of the cortical ER. This sorting to a subdomain of the ER abolishes proteasomal degradation and trafficking along the classical secretory (sec) pathway. In combination with the localization of IST2 mRNA to the bud tip and other redundant signals in Ist2, binding of PIPs leads to efficient accumulation of Ist2 at domains of the cortical ER from where the protein may reach the PM independently of the function of the sec-pathway.

A conserved, lipid-mediated sorting mechanism of yeast Ist2 and mammalian STIM proteins to the peripheral ER.

Sorting of yeast Ist2 to the plasma membrane (PM) or the cortical endoplasmic reticulum (ER) requires a cortical sorting signal (CSS(Ist2)) that interacts with lipids including phosphatidylinositol-4,5-bisphosphate (PI(4,5)P(2)) at the PM. Here, we show that the expression of Ist2 in mammalian cells resulted in a peripheral patch-like localization without any detection of Ist2 at the cell surface. Attached to C-termini of mammalian integral membrane proteins, the CSS(Ist2) targeted these proteins to PM-associated domains of the ER and abolished trafficking via the classical secretory pathway. The interaction of integral membrane proteins with PI(4,5)P(2) at the PM created ER-PM contacts. This process is similar to the regulated coupling of ER domains to the PM via stromal interaction molecule (STIM) proteins during store-operated Ca(2+) entry (SOCE). The CSS(Ist2) and the C-terminus of the ER-located Ca(2+) sensor STIM2 were sufficient to bind PI(4,5)P(2) and PI(3,4,5)P(3) at the PM, showing that an evolutionarily conserved mechanism is involved in the sorting of integral membrane proteins to PM-associated domains of the ER. Yeast Ist2 and STIM2 share a common basic and amphipathic signal at their extreme C-termini. STIM1 showed binding preference for liposomes containing PI(4,5)P(2), suggesting a specific contribution of lipids to the recruitment of ER domains to the PM during SOCE.

SEC18/NSF-independent, protein-sorting pathway from the yeast cortical ER to the plasma membrane.

Classic studies of temperature-sensitive secretory (sec) mutants have demonstrated that secreted and plasma membrane proteins follow a common SEC pathway via the endoplasmic reticulum (ER), Golgi apparatus, and secretory vesicles to the cell periphery. The yeast protein Ist2p, which is synthesized from a localized mRNA, travels from the ER to the plasma membrane via a novel route that operates independently of the formation of coat protein complex II-coated vesicles. In this study, we show that the COOH-terminal domain of Ist2p is necessary and sufficient to mediate SEC18-independent sorting when it is positioned at the COOH terminus of different integral membrane proteins and exposed to the cytoplasm. This domain functions as a dominant plasma membrane localization determinant that overrides other protein sorting signals. Based on these observations, we suggest a local synthesis of Ist2p at cortical ER sites, from where the protein is sorted by a novel mechanism to the plasma membrane.

A novel transport pathway for a yeast plasma membrane protein encoded by a localized mRNA.

Generally, plasma membrane (PM) proteins are cotranslationally inserted into the endoplasmic reticulum (ER) and travel in vesicles via the Golgi apparatus to the PM. In the yeast Saccharomyces cerevisiae, the polytopic membrane protein Ist2p is encoded by an mRNA that is localized to the cortex of daughter cells. It has been suggested that IST2 mRNA localization leads to the accumulation of the protein at the PM of daughter cells. Since small- and medium-sized daughter cells only contain cortical, but not perinuclear ER, this implies the local translation of Ist2p specifically at the cortical ER. Here, we show that localization of constitutively expressed IST2 mRNA is required for delivery of Ist2p to the PM of daughter, but not mother cells and that it does not result in daughter-specific Ist2p accumulation. In contrast to a PM-located hexose transporter (Hxt1p) that follows the standard secretory pathway, the trafficking of Ist2p is independent of myosin-mediated vesicular transport. Furthermore, colocalization experiments in mutants of the secretory pathway demonstrate that trafficking of Ist2p does not require the classical secretory machinery. These data suggest the existence of a novel trafficking pathway connecting specialized domains of the ER with the PM.

A complex peptide-sorting signal, but no mRNA signal, is required for the Sec-independent transport of Ist2 from the yeast ER to the plasma membrane.

The yeast integral plasma membrane protein Ist2 belongs to a group of membrane proteins which are synthesized from localized mRNAs. The protein reaches the plasma membrane via the ER on a route operating independently of the classical secretory pathway. We have identified a complex peptide-sorting signal located at the extreme C-terminus. This sorting signal operates independently of targeting information in IST2 mRNA and sorting to the plasma membrane does not require She-mediated mRNA transport into daughter cells. Based on these results, we suggest a posttranslational mechanism, which leads to the concentration of Ist2--via multimerization--at ER sites, followed by direct transport to the plasma membrane. This novel mechanism operates downstream of IST2 mRNA localization.

Yeast shuttling SR proteins Npl3p, Gbp2p, and Hrb1p are part of the translating mRNPs, and Npl3p can function as a translational repressor.

A major challenge in current molecular biology is to understand how sequential steps in gene expression are coupled. Recently, much attention has been focused on the linkage of transcription, processing, and mRNA export. Here we describe the cytoplasmic rearrangement for shuttling mRNA binding proteins in Saccharomyces cerevisiae during translation. While the bulk of Hrp1p, Nab2p, or Mex67p is not associated with polysome containing mRNAs, significant amounts of the serine/arginine (SR)-type shuttling mRNA binding proteins Npl3p, Gbp2p, and Hrb1p remain associated with the mRNA-protein complex during translation. Interestingly, a prolonged association of Npl3p with polysome containing mRNAs results in translational defects, indicating that Npl3p can function as a negative translational regulator. Consistent with this idea, a mutation in NPL3 that slows down translation suppresses growth defects caused by the presence of translation inhibitors or a mutation in eIF5A. Moreover, using sucrose density gradient analysis, we provide evidence that the import receptor Mtr10p, but not the SR protein kinase Sky1p, is involved in the timely regulated release of Npl3p from polysome-associated mRNAs. Together, these data shed light onto the transformation of an exporting to a translating mRNP.

Regulation of Adult CNS Axonal Regeneration by the Post-transcriptional Regulator Cpeb1.

Adult mammalian central nervous system (CNS) neurons are unable to regenerate following axonal injury, leading to permanent functional impairments. Yet, the reasons underlying this regeneration failure are not fully understood. Here, we studied the transcriptome and translatome shortly after spinal cord injury. Profiling of the total and ribosome-bound RNA in injured and naïve spinal cords identified a substantial post-transcriptional regulation of gene expression. In particular, transcripts associated with nervous system development were down-regulated in the total RNA fraction while remaining stably loaded onto ribosomes. Interestingly, motif association analysis of post-transcriptionally regulated transcripts identified the cytoplasmic polyadenylation element (CPE) as enriched in a subset of these transcripts that was more resistant to injury-induced reduction at the transcriptome level. Modulation of these transcripts by overexpression of the CPE binding protein, Cpeb1, in mouse and Drosophila CNS neurons promoted axonal regeneration following injury. Our study uncovered a global evolutionarily conserved post-transcriptional mechanism enhancing regeneration of injured CNS axons.

Asc1p, a WD40-domain containing adaptor protein, is required for the interaction of the RNA-binding protein Scp160p with polysomes.

Scp160p interacts in an mRNA-dependent manner with translating ribosomes via multiple RNA-binding heterogeneous nuclear ribonucleoprotein K-homology (KH) domains. In the present study, we show by protein-protein cross-linking that Scp160p is in close proximity to translation elongation factor 1A and the WD40 (Trp-Asp 40)-repeat containing protein Asc1p at ribosomes. Analysis of a truncation mutant revealed that the C-terminus of Scp160p is essential for ribosome binding and that Cys(1067) at the C-terminus of Scp160p is required to obtain these cross-links. The interaction of Scp160p with ribosomes depends on Asc1p. In fast-growing yeast cells, nearly all Asc1p is tightly bound to ribosomes, but it can also be present in a ribosome-free form depending on growth conditions. The functional homologue of Asc1p, mammalian RACK1 (receptor of activated C kinase), was previously characterized as an adaptor protein bridging activated signalling molecules with their substrates. Our results suggest that Scp160p connects specific mRNAs, ribosomes and a translation factor with an adaptor for signalling molecules. These interactions might regulate the translation activity of ribosomes programmed with specific mRNAs.

Ist2 in the yeast cortical endoplasmic reticulum promotes trafficking of the amino acid transporter Bap2 to the plasma membrane.

The equipment of the plasma membrane in Saccharomyces cerevisiae with specific nutrient transporters is highly regulated by transcription, translation and protein trafficking allowing growth in changing environments. The activity of these transporters depends on a H(+) gradient across the plasma membrane generated by the H(+)-ATPase Pma1. We found that the polytopic membrane protein Ist2 in the cortical endoplasmic reticulum (ER) is required for efficient leucine uptake during the transition from fermentation to respiration. Experiments employing tandem fluorescence timer protein tag showed that Ist2 was necessary for efficient trafficking of newly synthesized leucine transporter Bap2 from the ER to the plasma membrane. This finding explains the growth defect of ist2Δ mutants during nutritional challenges and illustrates the important role of physical coupling between cortical ER and plasma membrane.

Oligomerization and Ca2+/calmodulin control binding of the ER Ca2+-sensors STIM1 and STIM2 to plasma membrane lipids.

Ca2+ (calcium) homoeostasis and signalling rely on physical contacts between Ca2+ sensors in the ER (endoplasmic reticulum) and Ca2+ channels in the PM (plasma membrane). STIM1 (stromal interaction molecule 1) and STIM2 Ca2+ sensors oligomerize upon Ca2+ depletion in the ER lumen, contact phosphoinositides at the PM via their cytosolic lysine (K)-rich domains, and activate Ca2+ channels. Differential sensitivities of STIM1 and STIM2 towards ER luminal Ca2+ have been studied but responses towards elevated cytosolic Ca2+ concentration and the mechanism of lipid binding remain unclear. We found that tetramerization of the STIM1 K-rich domain is necessary for efficient binding to PI(4,5)P2-containing PM-like liposomes consistent with an oligomerization-driven STIM1 activation. In contrast, dimerization of STIM2 K-rich domain was sufficient for lipid binding. Furthermore, the K-rich domain of STIM2, but not of STIM1, forms an amphipathic α-helix. These distinct features of the STIM2 K-rich domain cause an increased affinity for PI(4,5)P2, consistent with the lower activation threshold of STIM2 and a function as regulator of basal Ca2+ levels. Concomitant with higher affinity for PM lipids, binding of CaM (calmodulin) inhibited the interaction of the STIM2 K-rich domain with liposomes in a Ca2+ and PI(4,5)P2 concentration-dependent manner. Therefore we suggest that elevated cytosolic Ca2+ concentration down-regulates STIM2-mediated ER-PM contacts via CaM binding.

Yeast Ist2 recruits the endoplasmic reticulum to the plasma membrane and creates a ribosome-free membrane microcompartment.

The endoplasmic reticulum (ER) forms contacts with the plasma membrane. These contacts are known to function in non-vesicular lipid transport and signaling. Ist2 resides in specific domains of the ER in Saccharomyces cerevisiae where it binds phosphoinositide lipids at the cytosolic face of the plasma membrane. Here, we report that Ist2 recruits domains of the yeast ER to the plasma membrane. Ist2 determines the amount of cortical ER present and the distance between the ER and the plasma membrane. Deletion of IST2 resulted in an increased distance between ER and plasma membrane and allowed access of ribosomes to the space between the two membranes. Cells that overexpress Ist2 showed an association of the nucleus with the plasma membrane. The morphology of the ER and yeast growth were sensitive to the abundance of Ist2. Moreover, Ist2-dependent effects on cytosolic pH and genetic interactions link Ist2 to the activity of the H(+) pump Pma1 in the plasma membrane during cellular adaptation to the growth phase of the culture. Consistently we found a partial colocalization of Ist2-containing cortical ER and Pma1-containing domains of the plasma membrane. Hence Ist2 may be critically positioned in domains that couple functions of the ER and the plasma membrane.

Role of endoplasmic reticulum domains in determining secretion routes.

Distinct domains of the endoplasmic reticulum (ER) can function as entry points into different protein-sorting routes. In addition to using the classical ER-Golgi pathway, one of these unconventional routes utilizes different combinations of machinery of the classical secretory pathway and components of the autophagosomal system.

A signal comprising a basic cluster and an amphipathic alpha-helix interacts with lipids and is required for the transport of Ist2 to the yeast cortical ER.

The yeast integral membrane protein Ist2 is encoded by a bud-localised mRNA and accumulates at patch-like domains of the cell periphery, either at the cortical ER or at ER-associated domains of the plasma membrane. Transport of IST2 mRNA and local protein synthesis are not prerequisite for this localisation, indicating that Ist2 can travel through the general ER to membranes at the cell periphery. Here, we describe that the accumulation of Ist2 at the cortical ER requires a cytosolically exposed complex sorting signal that can interact with lipids at the yeast plasma membrane. Binding of the Ist2 sorting signal to lipids and rapid and efficient transport of Ist2 from perinuclear to cortical ER depend on a cluster of lysine residues, the formation of an amphipathic alpha-helix and a patch of hydrophobic side chains positioned at one side of the amphipathic alpha-helix. We suggest that a direct interaction of the Ist2 sorting signal with lipids at the plasma membrane places Ist2 at contact sites between cortical ER and plasma membrane. This provides a physical link of an integral membrane protein of the cortical ER with the plasma membrane and might allow direct transport of proteins from cortical ER to domains of the plasma membrane.

The death receptor CD95 activates adult neural stem cells for working memory formation and brain repair.

Adult neurogenesis persists in the subventricular zone and the dentate gyrus and can be induced upon central nervous system injury. However, the final contribution of newborn neurons to neuronal networks is limited. Here we show that in neural stem cells, stimulation of the "death receptor" CD95 does not trigger apoptosis but unexpectedly leads to increased stem cell survival and neuronal specification. These effects are mediated via activation of the Src/PI3K/AKT/mTOR signaling pathway, ultimately leading to a global increase in protein translation. Induction of neurogenesis by CD95 was further confirmed in the ischemic CA1 region, in the naive dentate gyrus, and after forced expression of CD95L in the adult subventricular zone. Lack of hippocampal CD95 resulted in a reduction in neurogenesis and working memory deficits. Following global ischemia, CD95-mediated brain repair rescued behavioral impairment. Thus, we identify the CD95/CD95L system as an instructive signal for ongoing and injury-induced neurogenesis.

Unconventional mechanisms of protein transport to the cell surface of eukaryotic cells.

The classical secretion of soluble proteins and transport of integral membrane proteins to the cell surface require transit into and through the endoplasmic reticulum and the Golgi apparatus. Signal peptides or transmembrane domains target proteins for translocation into the lumen or insertion into the membrane of the endoplasmic reticulum, respectively. Here we discuss two mechanisms of unconventional protein targeting to plasma membranes, i.e., transport processes that are active in the absence of a functional Golgi system. We first focus on integral membrane proteins that are inserted into the endoplasmic reticulum but that, however, are transported to plasma membranes in a Golgi-independent manner. We then discuss soluble secretory proteins that are secreted from cells without any involvement of the endoplasmic reticulum and the Golgi apparatus.

The DEAD-box RNA helicase Dbp5 functions in translation termination.

In eukaryotes, termination of messenger RNA (mRNA) translation is mediated by the release factors eRF1 and eRF3. Using Saccharomyces cerevisiae as a model organism, we have identified a member of the DEAD-box protein (DBP) family, the DEAD-box RNA helicase and mRNA export factor Dbp5, as a player in translation termination. Dbp5 interacts genetically with both release factors and the polyadenlyate-binding protein Pab1. A physical interaction was specifically detected with eRF1. Moreover, we show that the helicase activity of Dbp5 is required for efficient stop-codon recognition, and intact Dbp5 is essential for recruitment of eRF3 into termination complexes. Therefore, Dbp5 controls the eRF3-eRF1 interaction and thus eRF3-mediated downstream events.