Secretory Membrane System and Golgi Apparatus

In Cell Biology (Third Edition), 2017

Membrane Bending Proteins

Proteins with BAR domains influence membrane trafficking by inducing membrane curvature, stabilizing curvature generated by other forces, and recruiting cytoplasmic proteins to membranes of a particular size or shape ( Fig. 21.15). Most BAR domains have a coiled-coil core of three α helices approximately 20 nm long. Classical BAR domains are shaped like a banana and are adapted to bind membranes with highly positive curvatures (ie, small vesicles). F-BAR and I-BAR domains fit on membranes with less-positive curvature or even on concave, negatively curved membranes. Most BAR domain proteins have at least one additional domain, such as an SH3 domain (see Fig. 25.10) that binds polyproline sequences in other proteins.

BAR domains use two mechanisms to bend membranes. In one, the intrinsic curvature of BAR domain dimers simply imposes its shape on the membrane substrate. In the second, amphipathic wedges present in the BAR domain insert into the bilayer, promoting membrane curvature by concerted displacement of lipids in the leaflet proximal to the site of insertion. For example, N-BARs have an N-terminal amphipathic helix within the BAR domain that can wedge into one leaflet of the bilayer to push the lipids apart. These two mechanisms are not mutually exclusive. The local deformations caused by insertion of the BAR domain can facilitate binding of additional BAR domains from other proteins and thereby generate a positive-feedback cycle for curvature propagation.

BAR domain proteins are important at membrane trafficking hubs where the generation of membrane curvature is coupled tightly to reorganization of the actin cytoskeleton and signaling through small GTPases. At the TGN, a BAR protein regulates biogenesis of transport carriers for regulated secretion. Other BAR proteins participate in clathrin-mediated endocytosis where they coordinate bud neck constriction, actin filament assembly and recruitment of proteins for fission and uncoating.

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Volume 2

Sonja Vermeren , ... Phillip T. Hawkins , in Handbook of Cell Signaling (Second Edition), 2010

DDEF/ASAP/AMAP/PAG/SHAG Arf GAPs

Members of this GAP family are characterized by an N-terminal BAR domain, PH and Arf GAP domains, ankyrin repeats, and a C-terminal extension comprising Pro-rich repeats and a SH3 domain. Both bind to a variety of other signaling proteins in vitro. To date, the in vivo function of ASAP/DDEF proteins is unclear. ASAP1/DDEF1 was originally purified from bovine brain as a PtdIns(4,5)P2-stimulated Arf1 GAP [26]. It has been found since that this catalytic stimulation depends on a conformational change induced by binding of PtdIns(4,5)P2 to ASAP1's PH domain [27, 28].

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Lipids

Belle Chang-Ileto , ... Gilbert Di Paolo , in Methods in Cell Biology, 2012

B Cells to Image Membrane Dynamics

COS7 cells are commonly used for the analysis of membrane tubulation caused by BAR domain-containing proteins ( Gallop et al., 2006; Itoh et al., 2005; Peter et al., 2004). These cells have a relatively flat morphology that allows for the visualization of membrane tubulation upon BAR-protein overexpression since a large number of tubular structures derived from the PM are in the same plane, particularly in the peripheral regions of the cell. Visualization of membrane tubules in other cell types, such as CHO and 293, was difficult if not unsuccessful.

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Organizational Cell Biology

L. Johannes , C. Wunder , in Encyclopedia of Cell Biology, 2016

Membrane Bending

The exact mechanism of membrane bending for retrograde trafficking from early endosomes is not known. However, different SNXs bearing BAR-domains and/or amphipathic helices were analyzed by in vitro studies (van Weering et al., 2012). Therefore, two SNX-driven mechanisms for membrane bending can be envisioned (Figure 4): (1) assymetric insertion of amphipathic helices and/or (2) scaffolding by BAR-domain containing SNXs. Whether SNXs are able to form scaffolds at physiological concentrations such as to drive membrane deformation (Sorre et al., 2012), still needs to be determined. Recently the concerted action of retromer SNXs and the WASH complex on tubule formation was supported by Seaman and colleagues (Freeman et al., 2014). This report indicated the binding of RME-8 to retromer SNXs and the WASH complex protein FAM21. Interestingly, clathrin could be localized to early endosomes (Popoff et al., 2009; Esk et al., 2010; Freeman et al., 2014), and RME-8 also binds to the clathrin uncoating ATPase Hsc70 (Girard et al., 2005). However, no reports currently exist on interactions between clathrin and components of the retromer machinery. Whether clathrin is directly involved in membrane deformation for other retrograde cargo protein requires further studies but seems likely when considering the function of the clathrin interactors AP1 (Meyer et al., 2000; Natsume et al., 2006), epsinR (Saint-Pol et al., 2004), and OCRL (Choudhury et al., 2005) in retrograde sorting on early endosomes.

Figure 4. Membrane bending mechanisms in relation to SNX proteins. (a) Insertion of helices leads to asymmetric expansion of the cytosolic leaflet and to subsequent membrane bending. (b) BAR domain binding can serve to recognize membrane curvature (low BAR-domain protein concentration on membranes), or to drive membrane bending through a scaffolding effect (high concentration).

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Organizational Cell Biology

M. Sharma , S. Caplan , in Encyclopedia of Cell Biology, 2016

Role of BAR Proteins in Regulating Signaling

While BAR domain-containing proteins are generally associated with regulating cytoskeletal dynamics and endocytic trafficking, a subgroup of BAR-domain proteins such as Fps/Fes (fps, Fujinami poultry sarcoma; fes, feline sarcoma) and Fer (Fes-related protein) play a crucial role in regulating signaling pathways. Fps/Fes and Fer are structurally unique members of the non-receptor protein tyrosine kinase family and were first isolated as oncogenes from avian and feline retroviruses ( Beemon, 1981). Subsequently, the cellular homolog of the Fps/Fes oncogene was identified in several species, including humans and mice, where they play key roles in inflammatory pathways, hemostasis, and innate immunity (Greer, 2002). Fes and Fer proteins contain an N-terminal F-BAR domain, an F-BAR extension (FX) domain that binds to phosphatidic acid, a SH2 domain that recognizes phosphotyrosine motifs, and a C-terminal kinase domain. Early studies on the viral Fps pointed to an important role of its N-terminal region in regulating transforming activity and membrane localization (Brooks-Wilson et al., 1989). Moreover, the F-BAR domains of Fes and Fer are necessary for their oligomerization, which potentiates their trans-autophosphorylation activity. However, whether oligomerization is required for their catalytic activity is not known (Greer, 2002). The Fes F-BAR domain can bind phosphoinositides and induce liposome tubulation in vitro (Zirngibl et al., 2001). The F-BAR domain in Fer interacts with the adherens-junction protein p120catenin, and overexpression of Fer results in increased phosphorylation of p120catenin and dissolution of adherens junctions and cell–cell adhesion (Piedra et al., 2003). The SH2 domain of Fps/Fes and Fer is thought to mediate phosphotyrosine-dependent interactions with several receptors such as EGFR and PDGFR, as well as signaling molecules such as PI3K and insulin receptor substrate-1 (Iwanishi et al., 2000). Moreover structural insights into the SH2 domain from Fes have suggested that the SH2 domain tightly interacts with the kinase domain, and activation of Fes kinase activity is closely coupled to SH2–kinase–ligand interactions (Filippakopoulos et al., 2008). The kinase domain of Fps/Fes phosphorylates, among other substrates, the protein BCR (breakpoint cluster region) (Maru et al., 1995), which has GEF activity toward Rho GTPase and RhoA and GAP activity toward Rac1, suggesting that Fps/Fes can regulate cytoskeletal dynamics by controlling BCR phosphorylation (Chuang et al., 1995). Other functions of Fes and Fer include vesicular trafficking and receptor internalization-mediated phosphorylation of protein substrates such as cortactin, platelet/endothelial cell adhesion molecule1 (Pecam-1), and β-catenin, as well as regulating cell migration, platelet activation, and leukocyte extravasation at sites of inflammation (Greer, 2002).

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The Molecular Biology of Cadherins

Jeff Hardin , ... Jonathan Pettitt , in Progress in Molecular Biology and Translational Science, 2013

4.6 SRGP-1/srGAP: Bending adhesions into shape

SRGP-1, the C. elegans Slit/Robo GAP homolog, is another lethal enhancer of hmp-1(fe4), and also enhances lethality in hmp-2(qm39) mutants. 75 Like its vertebrate counterparts, SRGP-1 contains an N-terminal F-BAR (Bin1, Amphiphysin, RVS167) domain, and a central GTPase activating (GAP) domain, which are thought to allow such proteins to regulate membrane curvature and modulate Rho family GTPase activity, respectively. 75 SRGP-1 colocalizes with the CCC at junctions; interestingly, expression of only the SRGP-1 F-BAR domain plus 200 downstream amino acids is sufficient to target SRGP-1 to junctions. This downstream sequence may be responsible for SRGP-1 interaction with the CCC as the F-BAR domain alone is not targeted to junctions. 75 SRGP-1 transgenes lacking GAP activity can rescue synergistic defects in hmp-2(qm39) mutants, suggesting that some of its functions are independent of its GAP activity. Whether SRGP-1's GAP activity is required in the more stringent hmp-1(fe4) background is currently unknown.

Overexpression of SRGP-1 leads to tubulations in the junctional membrane, which correlate with the level of SRGP-1 overexpression. Significantly, these tubulations contain HMR-1 and HMP-1, but not DLG-1, 75 suggesting that SRGP-1 aids adhesion by increasing the surface area of contact between adjacent cells at the level of the CCC. F-BAR proteins can also modulate actin dynamics more directly through their C-terminal domains 76 ; it remains unclear whether SRGP-1 has this capacity.

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AMPA Receptor Cell Biology/Trafficking

P.G.R. Hastie , J.M. Henley , in Encyclopedia of Neuroscience, 2009

Protein Interacting with C Kinase 1 Releases AMPARs from the Synapse

Protein interacting with C kinase (PICK1) interacts with a number of neuronal receptors, channels, and transporters. A single PDZ domain enables an interaction with short AMPARs leading to LTD. This occurs because PICK1 competes for the GRIP/ABP binding site on GluR2, uncoupling the AMPARs from the synaptic scaffold. This is controlled by protein kinase C (PKC) phosphorylation of GluR2 at Ser880, which prevents GRIP/ABP but not PICK1 binding. Phosphorylation is facilitated by PICK1 itself, which binds PKCα to target the kinase to AMPARs. PKCα binding removes an intramolecular interaction, exposing the BAR domain of PICK1. A second phosphorylation switch with the same function has recently been found at Tyr876, and this residue must be Src phosphorylated in response to drug treatment for internalization to occur. NSF hydrolysis of adenosine triphosphate (ATP) disrupts the PICK1–GluR2 interaction in complexes containing α-SNAP. This stabilizes the AMPARs at the surface. β-SNAP, however, prevents this dissociation and causes receptor internalization. Thus, in addition to actively inserting AMPARs at the synapse, NSF activity prevents their internalization. The presence of a BAR domain in PICK1 suggests this protein can recruit AMPARs to clathrin-coated vesicles (CCVs) in constitutive endocytosis in which AP2 does not seem to be involved.

Ca2+ binding to PICK1 increases the affinity of the GluR2 interaction. This has obvious implications for LTD, in which Ca2+ concentration is elevated in spines. The increase in affinity may be another switch in the balance between constitutive cycling and long-lasting endocytosis of AMPARs. PICK1 is not the only Ca2+-sensing molecule implicated in the endocytosis of AMPARs during LTD. The neuronal calcium-sensor hippocalcin binds AP2 in a Ca2+-dependent manner, and the Ca2+-sensing region of hippocalcin is required for the generation of LTD. It is thought that Ca2+ binding, which exposes a myristyl tail in hippocalcin, recruits AP2 to the membrane, enabling AMPAR sorting into CCVs. Hippocalcin also complexes with transferrin receptors but in a Ca2+-independent manner, suggesting the function of this protein is modified for a specific role in AMPAR endocytosis.

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Organizational Cell Biology

D.A. Sheffield , ... C.A. Mitchell , in Encyclopedia of Cell Biology, 2016

Retrograde Trafficking from Endosomes to Golgi

Retrograde transport is important for the retrieval of proteins and lipids back to the Golgi apparatus. Retrograde trafficking of cargo such as the mannose-6-phosphate receptor, which delivers hydrolases to endosomes before being recycled, occurs via the multimeric complex retromer (Bonifacino and Hurley, 2008). Additionally, there are retromer independent retrograde pathways, such as the transport of the plant toxin ricin (Cullen and Carlton, 2012). The yeast multimeric retromer complex consists of the trimer Vps26p–Vps29p–Vps35p and also Vps5p and Vps17p (Cullen and Carlton, 2012). In the mammalian complex there are two Vps5p homologues, SNX1 and SNX2 and two Vps17p homologues, SNX5 and SNX6 (Cullen and Carlton, 2012). Retrograde transport is achieved through tubule based sorting, whereby cargo is enriched in forming tubules which can then undergo fission for trafficking (Van Weering et al., 2012 ). A number of SNX proteins undergo coincidence detection via their phosphoinositide binding PX domain, and a membrane curvature-sensing BAR domain, targeted to the highly curved tubulovesicular endosomes where they are involved tubule formation ( Van Weering et al., 2012). The phosphoinositides involved in retrograde trafficking include PtdIns(3)P and PtdIns(3,5)P2, however, this remains an emerging field. In yeast, Vps34, the Class III PI3-kinase has a direct role in the regulation of retromer function (Burda et al., 2002). SNX1 and SNX2 localize to early endosomes, the site of PtdIns(3)P enrichment, and bind PtdIns(3)P and PtdIns(3,5)P2 (Carlton et al., 2005; Cozier et al., 2002). SNX3 a non-BAR domain containing SNX binds PtdIns(3)P and interacts with retromer in an alternative pathway for the recycling of Wntless (Harterink et al., 2011). The PIKfyve 5-kinase catalyzes the conversion of PtdIns(3)P to PtdIns(3,5)P2, and is necessary for normal retromer function, suggesting that turnover of PtdIns(3)P into PtdIns(3,5)P2 is an important requirement for retrograde trafficking (Rutherford et al., 2006). Consistent with this, the myotubularin 3-phosphatases, MTM-6 and MTM-9, which degrade PtdIns(3)P and PtdIns(3,5)P2 are required for Wnt signaling via retromer in C. elegans (Silhankova et al., 2010). The tubulation function of SNX1 is timed to Rab conversion in endosome maturation, and is most prominently observed on endosomes transitioning from Rab5 (early)-positive to Rab7 (late)-positive (Van Weering et al., 2012). Additionally, coincidence detection occurs between retromer, SNX3 and Rab7 on endosomes (Lorenowicz et al., 2014).

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International Review of Cell and Molecular Biology

J. Malinsky , M. Opekarová , in International Review of Cell and Molecular Biology, 2016

2.1.4 Endocytic Sites

Sites of clathrin-dependent endocytosis represent another important item on the list of the lateral compartments in the fungal plasma membrane. They are formed at the cytoplasmic side of the membrane and contain the proteins designated for internalization. The endocytic process requires strictly ordered recruitment of tens of different proteins. Using time-resolved electron tomography of yeast, a detailed 4D model including molecular details in a second time resolution could be reconstructed (Kukulski et al., 2012 ). The machinery of endocytic proteins includes early proteins, Ede1 and Syp1, determining the assembly of early, intermediate, and late coat proteins. Although clathrin associates with the flat membrane, it is not enough to initiate membrane curvature. Instead, the membrane is bent by polymerizing actin supported by BAR domain containing amphiphysins, which gradually extends the membrane invagination to a tubule ( Brach et al., 2014; Idrissi et al., 2008; Kukulski et al., 2012). High membrane curvature leads to the lateral segregation of specific lipids, which may also contribute to the vesicle scission. The newly formed vesicle is subsequently uncoated in cytoplasm by another set of well-described proteins (Goode et al., 2015; Weinberg and Drubin, 2012).

Little is known about the selection of the specific membrane area, in which the initiation of the new endocytic site occurs. The hypothesis that eisosomes provide the platform for endocytosis initiation (Walther et al., 2006) was ruled out by simultaneous localizations of fluorescently tagged MCC/eisosome residents Sur7 or Pil1 with early and late markers of endocytic sites, which revealed no overlap between the two structures (Brach et al., 2011; Grossmann et al., 2008; Seger et al., 2011)[Fig. 1(A, B)]. Moreover, a clear ultrastructural distinction between endocytic sites and eisosomes has been drawn by immunoelectron microscopy (Buser and Drubin, 2013). Another restriction for the formation of endocytic sites consists in the plasma membrane associated network of the peripheral ER. Mutual localization of early endocytic marker Ede1-GFP and cortical ER visualized by a luminal marker ss-dsRed-HDEL documented that the sites of endocytosis are distributed nonrandomly in the plasma membrane and restricted to areas free of cortical ER and MCC (Stradalova et al., 2012).

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Molecular Mechanisms of Memory

E. Marcora , ... M.B. Kennedy , in Learning and Memory: A Comprehensive Reference, 2008

4.32.3.2 GRIP/ABP, PICK1, and NSF

A distinct set of scaffold proteins bind selectively to GluR2/3 AMPARs and are believed to mediate regulation of their insertion and removal from synaptic sites (Lu and Ziff, 2005). The homologous PSD scaffold proteins termed GRIP (GRIP1) (Dong et al., 1997) and ABP (GRIP2) (Srivastava et al., 1998; Dong et al., 1999; Wyszynski et al., 1999) contain a total of six to seven PDZ domains and no obvious catalytic domain. The cytosolic tail of the GluR2 subunit of the AMPAR can bind to the fourth or fifth PDZ domain of either protein. GRIP/APBs exist in a palmitoylated form that is targeted to the postsynaptic membrane and a nonpalmitoylated form that is targeted to intracellular membranes (Yamazaki et al., 2001; DeSouza et al., 2002) and are thus thought to anchor AMPARs to either of these subcellular compartments in a regulated fashion to modulate AMPAR trafficking (Daw et al., 2000; Osten et al., 2000; Kim et al., 2001; Braithwaite et al., 2002; DeSouza et al., 2002; Fu et al., 2003; Hirbec et al., 2003; Seidenman et al., 2003). However, disruption of the interaction between the AMPAR and GRIP/ABP interferes most severely with the synaptic localization of the receptors (Dong et al., 1997).

PICK1 is a scaffold protein that contains a PDZ domain that binds selectively to protein kinase C-α (PKC-α) (Staudinger et al., 1997 ) and a BAR domain that that preferentially binds to curved membranes ( Peter et al., 2004). GRIP/ABP also associates with the cytosolic tail of the GluR2 and GluR3 subunits of the AMPA receptor via the single PDZ domain (Dev et al., 1999; Xia et al., 1999). Because PICK1 forms homomultimers, even when the PDZ domain is occupied, PICK1 is believed to bring PKC-α in proximity to the tail of GluR2 (Chung et al., 2000; Perez et al., 2001), where it can promote phosphorylation of GluR2 on serine 880 by PKC-α (Matsuda et al., 1999; Perez et al., 2001). Phosphorylation of GluR2 on serine 880 releases GluR2 from GRIP/APB but does not alter its association with PICK1.

PICK1 and GRIP/ABP appear to work together to regulate trafficking and surface expression of GluR2/3 receptors. Since GRIP/ABP serves as an anchor for GluR2 subunit-containing AMPARs on both postsynaptic and intracellular membrane compartments, dissociation of GluR2 from GRIP/ABP is required for both insertion and removal of AMPARs at synaptic sites (Daw et al., 2000; Osten et al., 2000; Kim et al., 2001; Braithwaite et al., 2002; Fu et al., 2003; Hirbec et al., 2003; Seidenman et al., 2003). PICK1 appears to promote dissociation of GluR2 from GRIP/ABP when it binds to the GRIP/ABP/GluR2 complex and induces phosphorylation of GluR2 on serine 880 by PKC-α. As GRIP/ABP unbinds from the phosphorylated form of GluR2, PDZ domains on PICK1 multimers can bind to GluR2. The BAR domain on PICK1 then promotes GluR2 association with budding endo- and exocytotic vesicles that mediate the trafficking of AMPARs to and from the postsynaptic membrane. Thus, PICK1 paradoxically promotes both internalization and recycling of GluR2 (Lu and Ziff, 2005). As predicted from these findings, disruption of interaction of GluR2/3 with PICK1 or GRIP/ABP impairs expression of LTD (Kim et al., 2001; Seidenman et al., 2003).

NSF (N-ethylmaleimide-sensitive fusion protein) and the clathrin adaptor AP2 interact with the cytosolic tail of the GluR2 subunit of the AMPAR in a membrane proximal region distinct from the C-terminal PDZ-binding domain that interacts with GRIP/ABP and PICK1. NSF and AP2 are also required for correct regulation of AMPAR trafficking and surface expression and consequently play important roles in synaptic plasticity, particularly LTD (Nishimune et al., 1998; Osten et al., 1998; Song et al., 1998; Luscher et al., 1999; Lee et al., 2002).

In addition to the AMPAR, GRIP/ABP binds to other synaptic proteins including the receptor tyrosine kinase EphB2 (Hoogenraad et al., 2005), the scaffold protein liprin-α (Wyszynski et al., 2002), and the neuronal Ras-GEF GRASP-1 (Ye et al., 2000). Interactions with these proteins likely help to regulate other, less well understood, aspects of AMPAR function.

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