Multiple and stepwise interactions between coatomer and ADP-ribosylation factor-1 (Arf1)-GTP

The small GTPase ADP-ribosylation factor-1 (Arf1) plays a key role in the formation of coat protein I (COP I)-coated vesicles. Upon recruitment to the donor Golgi membrane by interaction with dimeric p24 proteins, Arf1's GDP is exchanged for GTP. Arf1-GTP then dissociates from p24, and together with other Golgi membrane proteins, it recruits coatomer, the heptameric coat protein complex of COP I vesicles, from the cytosol. In this process, Arf1 was shown to specifically interact with the coatomer beta and gamma-COP subunits through its switch I region, and with epsilon-COP. Here, we mapped the interaction of the Arf1-GTP switch I region to the trunk domains of beta and gamma-COP. Site-directed photolabeling at position 167 in the C-terminal helix of Arf1 revealed a novel interaction with coatomer via a putative longin domain of delta-COP. Thus, coatomer is linked to the Golgi through multiple interfaces with membrane-bound Arf1-GTP. These interactions are located within the core, adaptor-like domain of coatomer, indicating an organizational similarity between the COP I coat and clathrin adaptor complexes.     

Distinct Actions of Rab3 and Rab27 GTPases on Late Stages of Exocytosis of Insulin

Rab GTPases associated with insulin‐containing secretory granules (SGs) are key in targeting, docking and assembly of molecular complexes governing pancreatic β‐cell exocytosis. Four Rab3 isoforms along with Rab27A are associated with insulin granules, yet elucidation of the distinct roles of these Rab families on exocytosis remains unclear. To define specific actions of these Rab families we employ Rab3GAP and/or EPI64A GTPase‐activating protein overexpression in β‐cells from wild‐type or Ashen mice to selectively transit the entire Rab3 family or Rab27A to a GDP‐bound state. Ashen mice carry a spontaneous mutation that eliminates Rab27A expression. Using membrane capacitance measurements we find that GTP/GDP nucleotide cycling of Rab27A is essential for generation of the functionally defined immediately releasable pool (IRP) and central to regulating the size of the readily releasable pool (RRP). By comparison, nucleotide cycling of Rab3 GTPases, but not of Rab27A, is essential for a kinetically rapid filling of the RRP with SGs. Aside from these distinct functions, Rab3 and Rab27A GTPases demonstrate considerable functional overlap in building the readily releasable granule pool. Hence, while Rab3 and Rab27A cooperate to generate release‐ready SGs in β‐cells, they also direct unique kinetic and functional properties of the exocytotic pathway.      

Binding with heat shock cognate protein HSC70 fine-tunes the Golgi association of the small GTPase ARL5B

ARL5B, an ARF-like small GTPase localized to the trans-Golgi, is known for regulating endosome-Golgi trafficking and promoting the migration and invasion of breast cancer cells. Although a few interacting partners have been identified, the mechanism of the shuttling of ARL5B between the Golgi membrane and the cytosol is still obscure. Here, using GFP-binding protein (GBP) pull-down followed by mass spectrometry, we identified heat shock cognate protein (HSC70) as an additional interacting partner of ARL5B. Our pull-down and isothermal titration calorimetry (ITC)-based studies suggested that HSC70 binds to ARL5B in an ADP-dependent manner. Additionally, we showed that the N-terminal helix and the nucleotide status of ARL5B contribute to its recognition by HSC70. The confocal microscopy and cell fractionation studies in MDA-MB-231 breast cancer cells revealed that the depletion of HSC70 reduces the localization of ARL5B to the Golgi. Using in vitro reconstitution approach, we provide evidence that HSC70 fine-tunes the association of ARL5B with Golgi membrane. Finally, we demonstrated that the interaction between ARL5B and HSC70 is important for the localization of cation independent mannose-6-phosphate receptor (CIMPR) at Golgi. Collectively, we propose a mechanism by which HSC70, a constitutively expressed chaperone, modulates the Golgi association of ARL5B, which in turn has implications for the Golgi-associated functions of this GTPase.     

Plant Sar1 isoforms with near-identical protein sequences exhibit different localisations and effects on secretion

In plants, differentiation of subdomains of the endoplasmic reticulum (ER) dedicated to protein export, the ER export sites (ERES), is influenced by the type of export-competent membrane cargo to be delivered to the Golgi. This raises a fundamental biological question: is the formation of transport intermediates at the ER for trafficking to the Golgi always regulated in the same manner? To test this, we followed the distribution and activity of two plant Sar1 isoforms. Sar1 is the small GTPase that regulates assembly of COPII (coat protein complex II) on carriers that transport secretory cargo from ER to Golgi. We show that, in contrast to a tobacco Sar1 isoform, the two Arabidopsis Sar1 GTPases were localised at ERES, independently of co-expression of Golgi-destined membrane cargo in tobacco cells. Although both isoforms labelled ERES, one was found to partition with the membrane fraction to a greater extent. The different distribution of fluorescent fusions of the two isoforms was influenced by the nature of an amino acid residue at the C-terminus of the protein, suggesting that the requirements for membrane association of the two GTPases are not equal. Furthermore, functional analyses based on the secretion of the bulk flow marker α-amylase indicated that over-expression of GTP-restricted mutants of the two isoforms caused different levels of ER export inhibition. These novel results indicate a functional heterogeneity among plant Sar1 isoforms.     

Role of the Lectin VIP36 in Post‐ER Quality Control of Human α1‐Antitrypsin

The leguminous‐type (L‐type) lectin VIP36 localizes to the Golgi apparatus and cycles early in the secretory pathway. In vitro, VIP36 binds high‐mannose glycans with a pH optimum of 6.5, a value similar to the luminal pH of the Golgi apparatus. Although the sugar‐binding properties of VIP36 in vitro have been characterized in detail, the function of VIP36 in the intact cell remains unclear as no convincing glycoprotein cargo has been identified. Here, we used yellow fluorescent protein (YFP) fragment complementation to identify luminal interaction partners of VIP36. By screening a human liver cDNA library, we identified the glycoprotein α1‐antitrypsin (α1‐AT) as a cargo of VIP36. The VIP36/α1‐AT complex localized to Golgi and endoplasmic reticulum (ER). In the living cell, VIP36 bound exclusively to the high‐mannose form of α1‐AT. The binding was increased when complex glycosylation was prevented by kifunensine and abolished when the glycosylation sites of α1‐AT were inactivated by mutagenesis. Silencing VIP36 accelerated α1‐AT transport, arguing against a role of VIP36 in anterograde traffic. The complex formed by VIP36 and α1‐AT in the Golgi recycled back to the ER. The combined data are most consistent with a function of VIP36 in post‐ER quality control of α1‐AT.     

Sterol transfer,PI4P consumption,and control of membrane lipid order by endogenous OSBP

The network of proteins that orchestrate the distribution of cholesterol among cellular organelles is not fully characterized. We previously proposed that oxysterol‐binding protein (OSBP) drives cholesterol/PI4P exchange at contact sites between the endoplasmic reticulum (ER) and the trans‐Golgi network (TGN). Using the inhibitor OSW‐1, we report here that the sole activity of endogenous OSBP makes a major contribution to cholesterol distribution, lipid order, and PI4P turnover in living cells. Blocking OSBP causes accumulation of sterols at ER/lipid droplets at the expense of TGN, thereby reducing the gradient of lipid order along the secretory pathway. OSBP consumes about half of the total cellular pool of PI4P, a consumption that depends on the amount of cholesterol to be transported. Inhibiting the spatially restricted PI4‐kinase PI4KIIIβ triggers large periodic traveling waves of PI4P across the TGN. These waves are cadenced by long‐range PI4P production by PI4KIIα and PI4P consumption by OSBP. Collectively, these data indicate a massive spatiotemporal coupling between cholesterol transport and PI4P turnover via OSBP and PI4‐kinases to control the lipid composition of subcellular membranes.     

Structure and function of the Lowe syndrome protein OCRL1

Oculocerebrorenal syndrome of Lowe (OCRL) is an X-linked disorder with the hallmark features of congenital cataracts, mental retardation and Fanconi syndrome of the kidney proximal tubules. OCRL was first described in 1952, and exactly four decades later, the gene responsible was identified and found to encode a protein highly homologous to inositol polyphosphate 5-phosphatase. This suggested that Lowe syndrome may represent an inborn error of inositol phosphate metabolism, and subsequent studies confirmed that such metabolism is indeed perturbed in Lowe syndrome cells. However, the mechanism by which loss of function of the OCRL1 protein brings about Lowe syndrome remains ill defined. In this review, I will discuss our understanding of OCRL1, including where it is localized, what it interacts with and what its possible functions might be. I will then discuss possible mechanisms by which loss of OCRL1 may bring about cellular defects that manifest themselves in the pathology of Lowe syndrome.     

Cryo‐EM structure of metazoan TRAPPIII,the multi‐subunit complex that activates the GTPase Rab1

The TRAPP complexes are nucleotide exchange factors that play essential roles in membrane traffic and autophagy. TRAPPII activates Rab11, and TRAPPIII activates Rab1, with the two complexes sharing a core of small subunits that affect nucleotide exchange but being distinguished by specific large subunits that are essential for activity in vivo. Crystal structures of core subunits have revealed the mechanism of Rab activation, but how the core and the large subunits assemble to form the complexes is unknown. We report a cryo‐EM structure of the entire Drosophila TRAPPIII complex. The TRAPPIII‐specific subunits TRAPPC8 and TRAPPC11 hold the catalytic core like a pair of tongs, with TRAPPC12 and TRAPPC13 positioned at the joint between them. TRAPPC2 and TRAPPC2L link the core to the two large arms, with the interfaces containing residues affected by disease‐causing mutations. The TRAPPC8 arm is positioned such that it would contact Rab1 that is bound to the core, indicating how the arm could determine the specificity of the complex. A lower resolution structure of TRAPPII shows a similar architecture and suggests that the TRAPP complexes evolved from a single ur‐TRAPP.     

Clathrin interactions with C-terminal regions of the yeast AP-1 beta and gamma subunits are important for AP-1 association with clathrin coats

Heterotetrameric adaptor (AP) complexes are thought to coordinate cargo recruitment and clathrin assembly during clathrin-coated vesicle biogenesis. We have identified, and characterized the physiological significance of clathrin-binding activities in the two large subunits of the AP-1 complex in Saccharomyces cerevisiae . Using GST-fusion chromatography, two clathrin-binding sites were defined in the β1 subunit that match consensus clathrin-binding sequences in other mammalian and yeast clathrin-binding proteins. Clathrin interactions were also identified with the C-terminal region of the γ subunit. When introduced into chromosomal genes, point mutations in the β1 clathrin-binding motifs, or deletion of the γ C-terminal region, reduced association of AP-1 with clathrin in coimmunoprecipitation assays. The β1 mutations or the γ truncation individually produced minor effects on AP-1 distribution by subcellular fractionation. However, when β1 and γ mutations were combined, severe defects were observed in AP-1 association with membranes and incorporation into clathrin-coated vesicles. The combination of subunit mutations accentuated growth and α-factor pheromone maturation defects in chc1-ts cells, though not to the extent caused by complete loss of AP-1 activity. Our results suggest that both the β1 and γ subunits contribute interactions with clathrin that are important for stable assembly of AP-1 complexes into clathrin coats in vivo .     

The chloroplast protein CPSAR1, dually localized in the stroma and the inner envelope membrane,is involved in thylakoid biogenesis

Thylakoid biogenesis is a crucial step for plant development involving the combined action of many cellular actors. CPSAR1 is shown here to be required for the normal organization of mature thylakoid stacks, and ultimately for embryo development. CPSAR1 is a chloroplast protein that has a dual localization in the stroma and the inner envelope membrane, according to microscopy studies and subfractionation analysis. CPSAR1 is close to the Obg nucleotide binding protein subfamily and displays GTPase activity, as demonstrated by in vitro assays. Disruption of the CPSAR1 gene via T‐DNA insertion results in the arrest of embryo development. In addition, transmission electron microscopy analysis indicates that mutant embryos are unable to develop thylakoid membranes, and remain white. Unstacked membrane structures resembling single lamellae accumulate in the stroma, and do not assemble into mature thylakoid stacks. CPSAR1 RNA interference induces partially developed thylakoids leading to pale‐green embryos. Altogether, the presented data demonstrate that CPSAR1 is a protein essential for the formation of normal thylakoid membranes, and suggest a possible involvement in the initiation of vesicles from the inner envelope membrane for the transfer of lipids to the thylakoids.     

Fluorescence approaches for determining protein conformations, interactions and mechanisms at membranes

Processes that occur at membranes are essential for the viability of every cell, but such processes are the least well understood at the molecular level. The complex nature and physical properties of the molecular components involved, as well as the requirement for two separated aqueous compartments, restrict the experimental approaches that can be successfully applied to examine the structure, conformational changes and interactions of the membrane-bound proteins that accomplish these processes. In particular, to accurately elucidate the molecular mechanisms that effect and regulate such processes, one must use experimental approaches that do not disrupt the structural integrity or functionality of the protein-membrane complexes being examined. To best accomplish this goal, especially when large multicomponent complexes and native membranes are involved, the optimal experimental approach to use is most often fluorescence spectroscopy. Using multiple independent fluorescence techniques, one can determine structural information in real time and in intact membranes under native conditions that cannot be obtained by crystallography, electron microscopy and NMR techniques, among others. Furthermore, fluorescence techniques provide a comprehensive range of information, from kinetic to thermodynamic, about the assembly, structure, function and regulation of membrane-bound proteins and complexes. This article describes the use of various fluorescence techniques to characterize different aspects of proteins bound to or embedded in membranes.     

Identification and characterization of AtCASP,a plant transmembrane Golgi matrix protein

Golgins are a family of coiled-coil proteins that are associated with the Golgi apparatus. They are necessary for tethering events in membrane fusion and may act as structural support for Golgi cisternae. Here we report on the identification of an Arabidopsis golgin which is a homologue of CASP, a known transmembrane mammalian and yeast golgin. Similar to its homologues, the plant CASP contains a long N-terminal coiled-coil region protruding into the cytosol and a C-terminal transmembrane domain with amino acid residues which are highly conserved across species. Through fluorescent protein tagging experiments, we show that plant CASP localizes at the plant Golgi apparatus and that the C-terminus of this protein is sufficient for its localization, as has been shown for its mammalian counterpart. In addition, we demonstrate that the plant CASP is able to localize at the mammalian Golgi apparatus. However, mutagenesis of a conserved tyrosine in the transmembrane domain revealed that it is necessary for ER export and Golgi localization of the Arabidopsis CASP in mammalian cells, but is not required for its correct localization in plant cells. These data suggest that mammalian and plant cells have different mechanisms for concentrating CASP in the Golgi apparatus.†These authors have contributed equally to the work     

Localization and control of expression of Nt-Syr1, a tobacco SNARE protein

Syntaxins and other SNARE proteins are crucial for intracellular vesicle trafficking, fusion and secretion. Previously, we isolated the syntaxin-related protein Nt-Syr1 from Nicotiana in a screen for ABA-related signalling elements, and demonstrated its role in determining the ABA sensitivity of stomatal guard cells. Because the location and expression of SNAREs are often important clues to their functioning, we have examined the distribution and stimulus-dependent expression of Nt-Syr1 between tissues, as well as its location within the cell, using antisera raised against purified recombinant peptides corresponding to overlapping cytosolic domains of Nt-Syr1. The Nt-Syr1 epitope was strongly represented in roots and to lesser extents in stems, leaves and flowers of well-watered plants. Biochemical analysis and examination of immunogold labelling under the electron microscope indicated Nt-Syr1 to be located primarily at the plasma membrane. Expression of the protein in leaves and to a lesser extent in flowers and stems was transiently enhanced by ABA, but not by auxin, kinetin or gibberellic acid. Expression in leaves was promoted by salt stress and wounding, but not by cold. By contrast, Nt-Syr1 levels in the root were unaffected by ABA. In the leaves, enhanced expression of Nt-Syr1 by salt stress was not observed in aba1 mutant Nicotiana, which is deficient in ABA synthesis, and in plants carrying the Arabidopsis abi1 transgene that suppresses a number of ABA-evoked responses in these plants. However, an enhanced expression in response to wounding was observed, even in the mutant backgrounds. We conclude that Nt-Syr1 expression at the plasma membrane is important for its function and is subject to control by parallel, stress-related signalling pathways, both dependent on and independent of ABA. Nt-Syr1 may be associated with additional functions, especially in the roots, that are unrelated to ABA or stress responses in the plant.     

A novel insertion mutation in atlastin 1 is associated with spastic quadriplegia,increased membrane tethering,and aberrant conformational switching

Hereditary spastic paraplegia (HSP) comprises a heterogeneous group of neuropathies affecting upper motor neurons and causing progressive gait disorder. Mutations in the gene SPG3A/atlastin-1 (ATL1), encoding a dynamin superfamily member, which utilizes the energy from GTP hydrolysis for membrane tethering and fusion to promote the formation of a highly branched, smooth endoplasmic reticulum (ER), account for approximately 10% of all HSP cases. The continued discovery and characterization of novel disease mutations are crucial for our understanding of HSP pathogenesis and potential treatments. Here, we report a novel disease-causing, in-frame insertion in the ATL1 gene, leading to inclusion of an additional asparagine residue at position 417 (N417ins). This mutation correlates with complex, early-onset spastic quadriplegia affecting all four extremities, generalized dystonia, and a thinning of the corpus callosum. We show using limited proteolysis and FRET-based studies that this novel insertion affects a region in the protein central to intramolecular interactions and GTPase-driven conformational change, and that this insertion mutation is associated with an aberrant prehydrolysis state. While GTPase activity remains unaffected by the insertion, membrane tethering is increased, indicative of a gain-of-function disease mechanism uncommon for ATL1-associated pathologies. In conclusion, our results identify a novel insertion mutation with altered membrane tethering activity that is associated with spastic quadriplegia, potentially uncovering a broad spectrum of molecular mechanisms that may affect neuronal function.     

Cell fractionation analysis of human CD8 glycoprotein transport between endoplasmic reticulum, intermediate compartment and Golgi complex in tissue cultured cells

We have set up an analytical cell fractionation procedure to dissect, by a non-morphological method, the anterograde transport of proteins from endoplasmic reticulum, intermediate compartment and Golgi complex in tissue cultured cells. Using this procedure after pulse-chase labelling of cells expressing human CD8 glycoprotein, we obtained results that: (1) support the view that the intermediate compartment is a distinct station in the export from the endoplasmic reticulum to the Golgi complex; and (2) strongly suggests that the O -glycosylation process starts after the intermediate compartment, presumably in the cis -Golgi complex.     

An extended tyrosine-targeting motif for endocytosis and recycling of the dense-core vesicle membrane protein phogrin

Integral membrane proteins of neuroendocine dense-core vesicles (DCV) appear to undergo multiple rounds of exocytosis; however, their trafficking and site of incorporation into nascent DCVs is unclear. Previous studies with phogrin (IA-2beta) identified sorting signals in the luminal domain that is cleaved post-translationally; we now describe an independent DCV targeting motif in the cytosolic domain that may function at the level of endocytosis and recycling. Pulse-chase radiolabeling and cell surface biotinylation experiments in the pituitary corticotroph cell line AtT20 showed that the mature 60/65 kDa form that resides in the DCV is generated by limited proteolysis in a post-trans Golgi network compartment with similar kinetics to the formation of the principal cargo, ACTH. Phogrin is exposed on the cell surface in response to stimuli and progressively internalized to a perinuclear compartment that overlaps with recycling endosomes marked by transferrin. Chimeric molecules of phogrin transmembrane and cytosolic sequences with the interleukin-2 receptor alpha chain (Tac) were sorted to DCVs through the action of an extended tyrosine-based motif Y(654)QELCRQRMA located in a 27aa sequence adjacent to the membrane-spanning domain. A 36aa domain terminating in this sequence conferred DCV localization to Tac in the absence of any other cytosolic or luminal phogrin components. The endocytosis and DCV targeting of phogrin Y(654) > A mutants correlated with the impaired binding of the phogrin cytosolic tail to the micro-subunit of the AP2 adaptor complex in vitro.     

High spatio-temporal resolution measurement of A1R and A2AR interactions combined with Iem-spFRET and E-FRET methods

A₁R-A_2AR heterodimers regulate striatal glutamatergic neurotransmission. However, few researches about kinetics have been reported. Here, we combined Iem-spFRET and E-FRET to investigate the kinetics of A₁R and A_2AR interaction. Iem-spFRET obtains the energy transfer efficiency of the whole cell. E-FRET gets energy transfer efficiency with high spatial resolution, whereas, it was prone to biases because background was easily selected due to manual operation. To study the interaction with high spatio-temporal resolution, Iem-spFRET was used to correct the deviation of E-FRET. In this paper, A₁R and A_2AR interaction was monitored, and the changes of FRET efficiency of the whole or/and partial cell membrane were described. The results showed that activation of A₁R or A_2AR leads to rapid aggregation, inhibition of A₁R or A_2AR leads to slow segregation, and the interaction is reversible. These results demonstrated that combination of Iem-spFRET and E-FRET could measure A₁R and A_2AR interaction with high spatio-temporal resolution.     

Multienzyme interactions of the de novo purine biosynthetic protein PAICS facilitate purinosome formation and metabolic channeling

There is growing evidence that mammalian cells deploy a mitochondria-associated metabolon called the purinosome to perform channeled de novo purine biosynthesis (DNPB). However, the molecular mechanisms of this substrate-channeling pathway are not well defined. Here, we present molecular evidence of protein–protein interactions (PPIs) between the human bifunctional phosphoribosylaminoimidazole carboxylase/succinocarboxamide synthetase (PAICS) and other known DNPB enzymes. We employed two orthogonal approaches: bimolecular fluorescence complementation, to probe PPIs inside live, intact cells, and co-immunoprecipitation using StrepTag-labeled PAICS that was reintegrated into the genome of PAICS-knockout HeLa cells (crPAICS). With the exception of amidophosphoribosyltransferase, the first enzyme of the DNPB pathway, we discovered PAICS interacts with all other known DNPB enzymes and with MTHFD1, an enzyme which supplies the 10-formyltetrahydrofolate cofactor essential for DNPB. We show these interactions are present in cells grown in both purine-depleted and purine-rich conditions, suggesting at least a partial assembly of these enzymes may be present regardless of the activity of the DNPB pathway. We also demonstrate that tagging of PAICS on its C terminus disrupts these interactions and that this disruption is correlated with disturbed DNPB activity. Finally, we show that crPAICS cells with reintegrated N-terminally tagged PAICS regained effective DNPB with metabolic signatures of channeled synthesis, whereas crPAICS cells that reintegrated C-terminally tagged PAICS exhibit reduced DNPB intermediate pools and a perturbed partitioning of inosine monophosphate into AMP and GMP. Our results provide molecular evidence in support of purinosomes and suggest perturbing PPIs between DNPB enzymes negatively impact metabolite flux through this important pathway.