The PHCCEx domain of Tiam1/2 is a novel protein‐ and membrane‐binding module

Tiam1 and Tiam2 (Tiam1/2) are guanine nucleotide‐exchange factors that possess the PH–CC–Ex (pleckstrin homology, coiled coil and extra) region that mediates binding to plasma membranes and signalling proteins in the activation of Rac GTPases. Crystal structures of the PH–CC–Ex regions revealed a single globular domain, PHCCEx domain, comprising a conventional PH subdomain associated with an antiparallel coiled coil of CC subdomain and a novel three‐helical globular Ex subdomain. The PH subdomain resembles the β‐spectrin PH domain, suggesting non‐canonical phosphatidylinositol binding. Mutational and binding studies indicated that CC and Ex subdomains form a positively charged surface for protein binding. We identified two unique acidic sequence motifs in Tiam1/2‐interacting proteins for binding to PHCCEx domain, Motif‐I in CD44 and ephrinB's and the NMDA receptor, and Motif‐II in Par3 and JIP2. Our results suggest the molecular basis by which the Tiam1/2 PHCCEx domain facilitates dual binding to membranes and signalling proteins.     

Coiled‐coil interactions are required for post‐Golgi R‐SNARE trafficking

The sorting of post‐Golgi R‐SNAREs (vesicle‐associated membrane protein (VAMP)1, 2, 3, 4, 7 and 8) is still poorly understood. To address this, we developed a system to investigate their localization, trafficking and cell‐surface levels. Here, we show that the distribution and internalization of VAMPs 3 and 8 are determined solely through a new conserved mechanism that uses coiled‐coil interactions, and that VAMP4 does not require these interactions for its trafficking. We propose that VAMPs 3 and 8 are trafficked while in a complex with Q‐SNAREs. We also show that the dileucine motif of VAMP4 is required for both its internalization and retrieval to the trans‐Golgi network. However, when the dileucine motif is mutated, the construct can still be internalized potentially through coiled‐coil interactions with Q‐SNAREs.     

Instability in a coiled‐coil signaling helix is conserved for signal transduction in soluble guanylyl cyclase

How nitric oxide (NO) activates its primary receptor, α1/β1 soluble guanylyl cyclase (sGC or GC‐1), remains unknown. Likewise, how stimulatory compounds enhance sGC activity is poorly understood, hampering development of new treatments for cardiovascular disease. NO binding to ferrous heme near the N‐terminus in sGC activates cyclase activity near the C‐terminus, yielding cGMP production and physiological response. CO binding can also stimulate sGC, but only weakly in the absence of stimulatory small‐molecule compounds, which together lead to full activation. How ligand binding enhances catalysis, however, has yet to be discovered. Here, using a truncated version of sGC from Manduca sexta, we demonstrate that the central coiled‐coil domain, the most highly conserved region of the ~150,000 Da protein, not only provides stability to the heterodimer but is also conformationally active in signal transduction. Sequence conservation in the coiled coil includes the expected heptad‐repeating pattern for coiled‐coil motifs, but also invariant positions that disfavor coiled‐coil stability. Full‐length coiled coil dampens CO affinity for heme, while shortening of the coiled coil leads to enhanced CO binding. Introducing double mutation αE447L/βE377L, predicted to replace two destabilizing glutamates with leucines, lowers CO binding affinity while increasing overall protein stability. Likewise, introduction of a disulfide bond into the coiled coil results in reduced CO affinity. Taken together, we demonstrate that the heme domain is greatly influenced by coiled‐coil conformation, suggesting communication between heme and catalytic domains is through the coiled coil. Highly conserved structural imperfections in the coiled coil provide needed flexibility for signal transduction.     

Interaction of Golgin‐84 with the COG Complex Mediates the Intra‐Golgi Retrograde Transport

The coiled‐coil Golgi membrane protein golgin‐84 functions as a tethering factor for coat protein I (COPI) vesicles. Protein interaction analyses have revealed that golgin‐84 interacts with another tether, the conserved oligomeric Golgi (COG) complex, through its subunit Cog7. Therefore, we explored the function of golgin‐84 as the tether for COPI vesicles of intra‐Golgi retrograde traffic. First, glycosylic maturation of both plasma membrane (CD44) and lysosomal (lamp1) glycoproteins was distorted in golgin‐84 knockdown (KD) cells. The depletion of golgin‐84 caused fragmentation of the Golgi with the mislocalization of Golgi resident proteins, resulting in the accumulation of vesicles carrying intra‐Golgi soluble N‐ethylmaleimide‐sensitive factor attachment protein receptors (SNAREs) and cis‐Golgi membrane protein GPP130. Similar observations were obtained by diminution of the COG complex, suggesting a strong correlation between the two tethers. Indeed, COG complex‐dependent (CCD) vesicles that accumulate in Cog3 or Cog7 KD cells carried golgin‐84. Surprisingly, the interaction between golgin‐84 and another candidate tethering partner CASP (CDP/cut alternatively spliced product) decreased in Cog3 KD cells. These results indicate that golgin‐84 on COPI vesicles interact with the COG complex before SNARE assembly, suggesting that the interaction of golgin‐84 with COG plays an important role in the tethering process of intra‐Golgi retrograde vesicle traffic.     

Targeted Disruption of an EH‐domain Protein Endocytic Complex,Pan1‐End3

Pan1 is a multi‐domain scaffold that enables dynamic interactions with both structural and regulatory components of the endocytic pathway. Pan1 is composed of Eps15 Homology (EH) domains which interact with adaptor proteins, a central region that is responsible for its oligomerization and C‐terminal binding sites for Arp2/3, F‐actin, and type‐I myosin motors. In this study, we have characterized the binding sites between Pan1 and its constitutive binding partner End3, another EH domain containing endocytic protein. The C‐terminal End3 Repeats of End3 associate with the N‐terminal part of Pan1's central coiled‐coil region. These repeats appear to act independently of one another as tandem, redundant binding sites for Pan1. The end3‐1 allele was sequenced, and corresponds to a C‐terminal truncation lacking the End3 Repeats. Mutations of the End3 Repeats highlight that those residues which are identical between these repeats serve as contact sites for the interaction with Pan1.      

Coiled‐coils: The long and short of it

Coiled‐coils are found in proteins throughout all three kingdoms of life. Coiled‐coil domains of some proteins are almost invariant in sequence and length, betraying a structural and functional role for amino acids along the entire length of the coiled‐coil. Other coiled‐coils are divergent in sequence, but conserved in length, thereby functioning as molecular spacers. In this capacity, coiled‐coil proteins influence the architecture of organelles such as centrioles and the Golgi, as well as permit the tethering of transport vesicles. Specialized coiled‐coils, such as those found in motor proteins, are capable of propagating conformational changes along their length that regulate cargo binding and motor processivity. Coiled‐coil domains have also been identified in enzymes, where they function as molecular rulers, positioning catalytic activities at fixed distances. Finally, while coiled‐coils have been extensively discussed for their potential to nucleate and scaffold large macromolecular complexes, structural evidence to substantiate this claim is relatively scarce.     

Control of RAB7 activity and localization through the retromer‐TBC1D5 complex enables RAB7‐dependent mitophagy

Retromer is an endosomal multi‐protein complex that organizes the endocytic recycling of a vast range of integral membrane proteins. Here, we establish an additional retromer function in controlling the activity and localization of the late endosomal small GTPase RAB7. Surprisingly, we found that RAB7 not only decorates late endosomes or lysosomes, but is also present on the endoplasmic reticulum, trans‐Golgi network, and mitochondrial membranes, a localization that is maintained by retromer and the retromer‐associated RAB7‐specific GAP TBC1D5. In the absence of either TBC1D5 or retromer, RAB7 activity state and localization are no longer controlled and hyperactivated RAB7 expands over the entire lysosomal domain. This lysosomal accumulation of hyperactivated RAB7 results in a striking loss of RAB7 mobility and overall depletion of the inactive RAB7 pool on endomembranes. Functionally, we establish that this control of RAB7 activity is not required for the recycling of retromer‐dependent cargoes, but instead enables the correct sorting of the autophagy related transmembrane protein ATG9a and autophagosome formation around damaged mitochondria during Parkin‐mediated mitophagy.     

Phosphatidylinositol 4‐phosphate on Rab7‐positive autophagosomes revealed by the freeze‐fracture replica labeling

Phosphatidylinositol 4‐phophate (PtdIns(4)P) is an essential signaling molecule in the Golgi body, endosomal system, and plasma membrane and functions in the regulation of membrane trafficking, cytoskeletal organization, lipid metabolism and signal transduction pathways, all mediated by direct interaction with PtdIns(4)P‐binding proteins. PtdIns(4)P was recently reported to have functional roles in autophagosome biogenesis. LC3 and GABARAP subfamilies and a small GTP‐binding protein, Rab7, are localized on autophagosomal membranes and participate at each stage of autophagosome formation and maturation. To better understand autophagosome biogenesis, it is essential to determine the localization of PtdIns(4)P and to examine its relationship with LC3 and GABARAP subfamilies and Rab7. To analyze PtdIns(4)P distribution, we used an electron microscopy technique that labels PtdIns(4)P on the freeze‐fracture replica of intracellular biological membranes, which minimizes the possibility of artificial perturbation because molecules in the membrane are physically immobilized in situ. Using this technique, we found that PtdIns(4)P is localized on the cytoplasmic, but not the luminal (exoplasmic), leaflet of the inner and outer membranes of autophagosomes. Double labeling revealed that PtdIns(4)P mostly colocalizes with Rab7, but not with LC3B, GABARAP, GABARAPL1 and GABARAPL2. Rab7 plays essential roles in autophagosome maturation and in autophagosome‐lysosome fusion events. We suggest that PtdIns(4)P is localized to the cytoplasmic leaflet of the autophagosome at later stages, which may illuminate the importance of PtdIns(4)P at the later stages of autophagosome formation.      

GIT1 is a novel MEK1–ERK1/2 scaffold that localizes to focal adhesions

Cell polarity is critical for cell migration and requires localized signal transduction in subcellular domains. Recent evidence demonstrates that activation of ERK1/2 (extracellular‐signal‐regulated kinase 1/2) in focal adhesions is essential for cell migration. GIT1 (G‐protein‐coupled receptor kinase‐interacting protein 1) has been shown to bind paxillin and regulate focal‐adhesion disassembly. We have previously reported that GIT1 binds to MEK1 [MAPK (mitogen‐activated protein kinase)/ERK kinase 1] and acts as a scaffold to enhance ERK1/2 activation in response to EGF (epidermal growth factor). In the present study we show that GIT1 associates with ERK1/2 in focal adhesions and this association increases after EGF stimulation. The CC (coiled‐coil) domain of ERK1/2 is required for association with GIT1, translocation to focal adhesions, and cell spreading and migration. Immunofluorescent staining showed that, after EGF stimulation, GIT1 co‐localized with pERK1/2 (phosphorylated ERK1/2) in focal adhesions. The binding of GIT1 and ERK1/2 was functionally important, since transfecting an ERK2 mutant lacking the CC domain [ERK2(del CC)] significantly decreased pERK1/2 translocation to focal adhesions, cell spreading and migration induced by EGF. In summary, the CC domain of ERK1/2 is necessary for binding to GIT1, for ERK1/2 activation in focal adhesions, and for cell spreading and migration.     

Oxysterol‐binding protein recruitment and activity at the endoplasmic reticulum‐Golgi interface are independent of Sac1

Oxysterol‐binding protein (OSBP) localizes to endoplasmic reticulum (ER)‐Golgi contact sites where it transports cholesterol and phosphatidylinositol 4‐phosphate (PI‐4P), and activates lipid transport and biosynthetic activities. The PI‐4P phosphatase Sac1 cycles between the ER and Golgi apparatus where it potentially regulates OSBP activity. Here we examined whether the ER‐Golgi distribution of endogenous or ectopically expressed Sac1 influences OSBP activity. OSBP and Sac1 co‐localized at apparent ER‐Golgi contact sites in response to 25‐hydroxycholesterol (25OH), cholesterol depletion and p38 MAPK inhibitors. A Sac1 mutant that is unable to exit the ER did not localize with OSBP, suggesting that sterol perturbations cause Sac1 transport to the Golgi apparatus. Ectopic expression of Sac1 in the ER or Golgi apparatus, or Sac1 silencing, did not affect OSBP localization to ER‐Golgi contact sites, OSBP‐dependent activation of sphingomyelin synthesis, or cholesterol esterification in the ER. p38 MAPK inhibition and retention of Sac1 in the Golgi apparatus also caused OSBP phosphorylation and OSBP‐dependent activation of sphingomyelin synthesis at ER‐Golgi contacts. These results demonstrate that Sac1 expression in either the ER or Golgi apparatus has a minimal impact on the PI‐4P that regulates OSBP activity or recruitment to contact sites.      

Association of a novel PDZ domain-containing peripheral Golgi protein with the Q-SNARE (Q-soluble N-ethylmaleimide-sensitive fusion protein (NSF) attachment protein receptor) protein syntaxin 6

PDZ domains are involved in the scaffolding and assembly of multi-protein complexes at various subcellular sites. We describe here the isolation and characterization of a novel PDZ domain-containing protein that localizes to the Golgi apparatus. Using an in silico cloning approach, we have identified and isolated a cDNA encoding a ubiquitously expressed 59-kDa protein that we call FIG. It is composed of two coiled coil regions, a leucine zipper, and a single PDZ domain. Cytological studies using indirect immunofluorescence microscopy revealed that FIG is a peripheral protein that uses one of its coiled coil domains to localize to the Golgi apparatus. To ascertain the modalities of this Golgi localization, the same coiled coil region was tested for its ability to interact with a panel of coiled coil domain-containing integral membrane Golgi proteins. Using a series of GST fusion protein binding assays, co-immunofluorescence and co-immunoprecipitation experiments, we show that FIG specifically binds to the coiled coil domain-containing Q-SNARE (Q-soluble NSF attachment protein receptor) protein syntaxin 6 both in vitro and in vivo. The structural features of FIG and its interaction with a SNARE protein suggest that FIG may play a role in membrane vesicle trafficking. This is the first example of a PDZ domain-containing peripheral protein that localizes to the Golgi through a coiled coil-mediated interaction with a resident membrane protein. Our results broaden the scope of PDZ domain-mediated functions.     

Structure of the WD40‐domain of human ATG16L1

Autophagy‐related protein ATG16L1 is a component of the mammalian ATG12～ATG5/ATG16L1 complex, which acts as E3‐ligase to catalyze lipidation of LC3 during autophagosome biogenesis. The N‐terminal part of ATG16L1 comprises the ATG5‐binding site and coiled‐coil dimerization domain, both also present in yeast ATG16 and essential for bulk and starvation induced autophagy. While absent in yeast ATG16, mammalian ATG16L1 further contains a predicted C‐terminal WD40‐domain, which has been shown to be involved in mediating interaction with diverse factors in the context of alternative functions of autophagy, such as inflammatory control and xenophagy. In this work, we provide detailed information on the domain boundaries of the WD40‐domain of human ATG16L1 and present its crystal structure at a resolution of 1.55 Å.     

ProP‐ProP and ProP‐phospholipid interactions determine the subcellular distribution of osmosensing transporter ProP in Escherichia coli

Osmosensing transporter ProP protects bacteria from osmotically induced dehydration by mediating the uptake of zwitterionic osmolytes. ProP activity is a sigmoidal function of the osmolality. ProP orthologues share an extended, cytoplasmic C‐terminal domain. Orthologues with and without a C‐terminal, α‐helical coiled‐coil domain respond similarly to the osmolality. ProP concentrates at the poles and septa of Escherichia coli cells in a cardiolipin (CL)‐dependent manner. The roles of phospholipids and the C‐terminal domain in subcellular localization of ProP were explored. Liposome association of peptides representing the C‐terminal domains of ProP orthologues and variants in vitro was compared with subcellular localization of the corresponding orthologues and variants in vivo. In the absence of coiled‐coil formation, the C‐terminal domain bound liposomes and ProP concentrated at the cell poles in a CL‐independent manner. The presence of the coiled‐coil replaced those phenomena with CL‐dependent binding and localization. The effects of amino acid replacements on lipid association of the C‐terminal peptide fully recapitulated their effects on the subcellular localization of ProP. These data suggest that polar localization of ProP results from association of its C‐terminal domain with the anionic lipid‐enriched membrane at the cell poles. The coiled‐coil domain present on only some orthologues renders that phenomenon CL‐dependent.     

The DNA‐ and RNA‐binding protein FACTOR of DNA METHYLATION 1 requires XH domain‐mediated complex formation for its function in RNA‐directed DNA methylation

Studies have identified a sub‐group of SGS3‐LIKE proteins including FDM1–5 and IDN2 as key components of RNA‐directed DNA methylation pathway (RdDM). Although FDM1 and IDN2 bind RNAs with 5′ overhangs, their functions in the RdDM pathway remain to be examined. Here we show that FDM1 interacts with itself and with IDN2. Gel filtration suggests that FDM1 may exist as a homodimer in a heterotetramer complex in vivo. The XH domain of FDM1 mediates the FDM1–FDM1 and FDM1–IDN2 interactions. Deletion of the XH domain disrupts FDM1 complex formation and results in loss‐of‐function of FDM1. These results demonstrate that XH domain‐mediated complex formation of FDM1 is required for its function in RdDM. In addition, FDM1 binds unmethylated but not methylated DNAs through its coiled‐coil domain. RNAs with 5′ overhangs does not compete with DNA for binding by FDM1, indicating that FDM1 may bind DNA and RNA simultaneously. These results provide insight into how FDM1 functions in RdDM.     

Structural insights into Acyl‐coenzyme A binding domain containing 3 (ACBD3) protein hijacking by picornaviruses

Many picornaviruses hijack the Golgi resident Acyl‐coenzyme A binding domain containing 3 (ACBD3) protein in order to recruit the phosphatidylinositol 4‐kinase B (PI4KB) to viral replication organelles (ROs). PI4KB, once recruited and activated by ACBD3 protein, produces the lipid phosphatidylinositol 4‐phosphate (PI4P), which is a key step in the biogenesis of viral ROs. To do so, picornaviruses use their small nonstructural protein 3A that binds the Golgi dynamics domain of the ACBD3 protein. Here, we present the analysis of the highly flexible ACBD3 proteins and the viral 3A protein in solution using small‐angle X‐ray scattering and computer simulations. Our analysis revealed that both the ACBD3 protein and the 3A:ACBD3 protein complex have an extended and flexible conformation in solution.     

Ethanol stimulates the in vivo axonal movement of neuropeptide dense‐core vesicles in Drosophila motor neurons

Proper neuronal function requires essential biological cargoes to be packaged within membranous vesicles and transported, intracellularly, through the extensive outgrowth of axonal and dendritic fibers. The precise spatiotemporal movement of these cargoes is vital for neuronal survival and, thus, is highly regulated. In this study we test how the axonal movement of a neuropeptide‐containing dense‐core vesicle (DCV ) responds to alcohol stressors. We found that ethanol induces a strong anterograde bias in vesicle movement. Low doses of ethanol stimulate the anterograde movement of neuropeptide‐DCV while high doses inhibit bi‐directional movement. This process required the presence of functional kinesin‐1 motors as reduction in kinesin prevented the ethanol‐induced stimulation of the anterograde movement of neuropeptide‐DCV . Furthermore, expression of inactive glycogen synthase kinase 3 (GSK ‐3β) also prevented ethanol‐induced stimulation of neuropeptide‐DCV movement, similar to pharmacological inhibition of GSK ‐3β with lithium. Conversely, inhibition of PI 3K/AKT signaling with wortmannin led to a partial prevention of ethanol‐stimulated transport of neuropeptide‐DCV . Taken together, we conclude that GSK ‐3β signaling mediates the stimulatory effects of ethanol. Therefore, our study provides new insight into the physiological response of the axonal movement of neuropeptide‐DCV to exogenous stressors.

Cover Image for this Issue: doi: 10.1111/jnc.14165 .

     

SAS‐6 coiled‐coil structure and interaction with SAS‐5 suggest a regulatory mechanism in C. elegans centriole assembly

The centriole is a conserved microtubule‐based organelle essential for both centrosome formation and cilium biogenesis. Five conserved proteins for centriole duplication have been identified. Two of them, SAS‐5 and SAS‐6, physically interact with each other and are codependent for their targeting to procentrioles. However, it remains unclear how these two proteins interact at the molecular level. Here, we demonstrate that the short SAS‐5 C‐terminal domain (residues 390–404) specifically binds to a narrow central region (residues 275–288) of the SAS‐6 coiled coil. This was supported by the crystal structure of the SAS‐6 coiled‐coil domain (CCD), which, together with mutagenesis studies, indicated that the association is mediated by synergistic hydrophobic and electrostatic interactions. The crystal structure also shows a periodic charge pattern along the SAS‐6 CCD, which gives rise to an anti‐parallel tetramer. Overall, our findings establish the molecular basis of the specific interaction between SAS‐5 and SAS‐6, and suggest that both proteins individually adopt an oligomeric conformation that is disrupted upon the formation of the hetero‐complex to facilitate the correct assembly of the nine‐fold symmetric centriole.     

A conserved sequence in calmodulin regulated spectrin‐associated protein 1 links its interaction with spectrin and calmodulin to neurite outgrowth

Calmodulin regulated spectrin‐associated protein 1 (CAMSAP1) is a vertebrate microtubule‐binding protein, and a representative of a family of cytoskeletal proteins that arose with animals. We reported previously that the central region of the protein, which contains no recognized functional domain, inhibited neurite outgrowth when over‐expressed in PC12 cells [Baines et al., Mol. Biol. Evol. 26 (2009), p. 2005]. The CKK domain (DUF1781) binds microtubules and defines the CAMSAP/ssp4 family of animal proteins (Baines et al. 2009). In the central region, three short well‐conserved regions are characteristic of CAMSAP‐family members. One of these, CAMSAP‐conserved region 1 (CC1), bound to both βIIΣ1‐spectrin and Ca²⁺/calmodulin in vitro. The binding of Ca²⁺/calmodulin inhibited spectrin binding. Transient expression of CC1 in PC12 cells inhibited neurite outgrowth. siRNA knockdown of CAMSAP1 inhibited neurite outgrowth in PC12 cells or primary cerebellar granule cells: this could be rescued in PC12 cells by wild‐type CAMSAP1‐enhanced green fluorescent protein, but not by a CC1 mutant. We conclude that CC1 represents a functional region of CAMSAP1, which links spectrin‐binding to neurite outgrowth.