Membrane Sorting Research Articles

The HUS‐box is required for allosteric regulation of the Sec7 Arf‐GEF

The Golgi complex is the central membrane and protein sorting station of eukaryotic cells. Activation of Arf GTPases is essential for vesicle formation via recruitment of cargo adaptors and coat proteins necessary for Golgi trafficking. Arf activation is spatially and temporally regulated by distinct guanine nucleotide exchange factors (GEFs) at different Golgi compartments. The yeast Arf‐GEF Sec7 is a conserved and essential activator of Arf1 at the trans‐Golgi network. Sec7 contains a highly conserved regulatory region, the HUS‐box, with an unknown mechanistic role. In this study we explore how the HUS‐box, which is N‐terminal to the catalytic domain, acts together with C‐terminal regulatory domains in the allosteric activation of Sec7. We show that mutation of the HUS‐box disturbs positive feedback and allosteric activation of Sec7 by the GTPase Ypt31, the yeast Rab11 homolog. Taken together, our data lead to a model in which the inter‐ and intramolecular interactions of the HUS‐box and C‐terminus are necessary for the allosteric activation of Sec7.Support or Funding InformationThis work was supported by NIH/NIGMS award R01GM09861 to J.C.F. and NIH/NIGMS training grant T32GM007273 to S.L.H.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

A subunit of the HOPS endocytic tethering complex, FgVps41, is important for fungal development and plant infection in Fusarium graminearum.

The signals by which eukaryotic cells communicate with the environment are usually mediated by vesicle trafficking to be attenuated or terminated. However, vesicle trafficking-mediated signal transmission during interactions between pathogens and host plants is poorly understood. Here, we identified and characterized the vacuole sorting protein FgVps41, which is the yeast HOPS tethering complex subunit Vps41 homolog in Fusarium graminearum. Targeted gene deletion demonstrated that FgVps41 is important forvegetative growth, asexual/sexual development, conidial morphology, plant infection and deoxynivalenol production. Cellular localization and cytological examinations revealed that FgVps41 localizes to early/late endosomes and vacuole membrane, and is recruited to prevacuolar compartments and vacuole membrane by interacting with FgRab7 in F. graminearum. Furthermore, we found FgVps41 mediates vacuole membrane fusion and sorting of FgApeI, a cargo protein involving in the cytosol-to-vacuole targeting pathway. In addition, we found that FgVps41 interacts with FgYck3, a vacuolar type I casein kinase, which regulates vesicle fusion in the AP-3 pathway. Deletion of FgYck3 showed similar phenotypes to the ΔFgvps41 mutant, and both FgRab7 and FgYck3 regulate the normal localization of FgVps41. Collectively, our results demonstrate that FgVps41 acts as a HOPS tethering complex subunit and is important for the development of infection-related morphogenesis in F. graminearum.

Molecular Mechanism of Microdomain Dependent Protein Trafficking

Eukaryotic cells are organized into spatially and functionally distinct membrane-bound organelles, whose functions are defined by their composition. Accurate sorting of membrane components between these compartments is necessary for maintaining organelle identity. For most membrane proteins, the determinants of their steady-state subcellular localization remain unknown. Lateral membrane domains (lipid rafts) provide an ideal platform for membrane sorting processes. However, the structural determinants of protein association with such domains are almost entirely unknown. We developed and characterized a robust experimental system for quantitative measurements of raft affinity in intact plasma membranes and used it to explore the determinants of transmembrane protein recruitment into raft domains. We identified several structural features associated with raft affinity, and established a quantitative and functional relationship between raft association and subcellular protein localization. Specifically, we observed that raft association is fully sufficient for PM recycling of certain proteins, and that abrogation of raft partitioning for these proteins led to their degradation in the lysosomes. These findings support the conclusion that ordered membrane domains mediate recycling of specific membrane components from the endosomal compartments to the PM. We have proceeded to define the molecular machinery that mediates raft lipid and protein sorting and recycling to the PM. Using a set of orthogonal transmembrane proteins as probes of raft and non-raft domains, we developed a high throughput siRNA screen to dissect the molecular machinery and dynamics for raft-mediated sorting. We identified a number of validated hits including known players of the early endocytic traffic (Rab5 and EEA1), but also novel players that appear to define a distinct class of trafficking mediators specific for raft-associated proteins. This pathway is not dependent on the classical recycling pathways defined by Rab4 and Rab11, rather defining a novel route for PM recycling of raft-preferring cargo.

Polarity of Neuronal Membrane Traffic Requires Sorting of Kinesin Motorcargo During Entry into Dendrites by a Microtubule-Associated Septin

Neuronal function requires axon-dendrite membrane polarity, which depends on sorting of membrane traffic during entry into axons. Due to a microtubule network of mixed polarity, dendrites receive vesicles from the cell body without apparent capacity for directional sorting. We found that in the initial segments of dendrites, axonally-destined cargos move with a retrograde bias toward the cell body, while dendritically-destined cargos are biased in the anterograde direction. A microtubule-associated septin (SEPT9), which localizes specifically in dendrites, impedes axonal cargo of kinesin-1/KIF5 and boosts kinesin-3/KIF1 motor-cargo further into dendrites. In neurons and in vitro single-molecule motility assays, SEPT9 suppresses kinesin-1/KIF5 and enhances kinesin-3/KIF1 in a manner that depends on a lysinerich loop of the kinesin motor domain. This differential regulation impacts partitioning of neuronal membrane proteins into axonsdendrites. Thus, polarized membrane traffic requires sorting during entry into dendrites by a septin-mediated mechanism that bestows directional bias on microtubules of mixed orientation.

Identification and characterization of the mitochondrial membrane sorting signals in phosphatidylserine decarboxylase 1 from Saccharomyces cerevisiae

Phosphatidylserine decarboxylase 1 (Psd1p) catalyzes the formation of the majority of phosphatidylethanolamine (PE) in the yeast Saccharomyces cerevisiae. Psd1p is localized to mitochondria, anchored to the inner mitochondrial membrane (IMM) through membrane spanning domains and oriented towards the mitochondrial intermembrane space. We found that Psd1p harbors at least two inner membrane-associated domains, which we named IM1 and IM2. IM1 is important for proper orientation of Psd1p within the IMM (Horvath et al., J. Biol. Chem. 287 (2012) 36744–55), whereas it remained unclear whether IM2 is important for membrane-association of Psd1p. To discover the role of IM2 in Psd1p import, processing and assembly into the mitochondria, we constructed Psd1p variants with deletions in IM2. Removal of the complete IM2 led to an altered topology of the protein with the soluble domain exposed to the matrix and to decreased enzyme activity. Psd1p variants lacking portions of the N-terminal moiety of IM2 were inserted into IMM with an altered topology. Psd1p variants with deletions of C-terminal portions of IM2 accumulated at the outer mitochondrial membrane and lost their enzyme activity. In conclusion we showed that IM2 is essential for full enzymatic activity, maturation and correct integration of yeast Psd1p into the inner mitochondrial membrane.

Structural determinants and functional consequences of protein affinity for membrane rafts

Eukaryotic plasma membranes are compartmentalized into functional lateral domains, including lipid-driven membrane rafts. Rafts are involved in most plasma membrane functions by selective recruitment and retention of specific proteins. However, the structural determinants of transmembrane protein partitioning to raft domains are not fully understood. Hypothesizing that protein transmembrane domains (TMDs) determine raft association, here we directly quantify raft affinity for dozens of TMDs. We identify three physical features that independently affect raft partitioning, namely TMD surface area, length, and palmitoylation. We rationalize these findings into a mechanistic, physical model that predicts raft affinity from the protein sequence. Application of these concepts to the human proteome reveals that plasma membrane proteins have higher raft affinity than those of intracellular membranes, consistent with raft-mediated plasma membrane sorting. Overall, our experimental observations and physical model establish general rules for raft partitioning of TMDs and support the central role of rafts in membrane traffic.

The polymorphism L204F affects transport and membrane expression of the sodium-dependent organic anion transporter SOAT (SLC10A6)

Sodium-dependent organic anion transporter (SOAT) represents a membrane transporter specific for sulfated steroid hormones, which are supposed to participate in the regulation of reproductive processes. In man, SOAT shows predominant mRNA expression in the testis and here was localized to primary spermatocytes. SOAT mRNA expression is significantly downregulated in different disorders of spermatogenesis, including hypospermatogenesis. The resulting decline of SOAT-mediated transport of sulfated steroids may participate in the impairment of functional spermatogenesis. Apart from downregulation of SOAT mRNA expression, genetic polymorphisms affecting the transport function of SOAT may have the same negative effect on spermatogenesis. Therefore, in the present study we searched for functionally relevant SOAT polymorphisms, aiming to comparatively analyze their occurrence in patients with impaired spermatogenesis vs. patients with intact spermatogenesis. We found that the SOAT polymorphism L204F showed a significantly reduced transport function for DHEAS when expressed in HEK293 cells. Although the Km value was identical with that of the SOAT wildtype, the Vmax value dramatically declined for the SOAT-L204F variant (942.5 vs. 313.6pmol×mg protein−1×min−1). Although the same amount of total SOAT-L204F protein was detected in transfected HEK293 cells compared to the SOAT wildtype, plasma membrane expression was significantly reduced, which points to a plasma membrane sorting defect of the SOAT-L204F variant. Groups of 20 subjects with normal spermatogenesis and 26 subjects with hypospermatogenesis were genotyped for this polymorphism. Both groups showed nearly identical distributions of the SOAT-L204F polymorphism (∼10% heterozygous and ∼5% homozygous), indicating that this polymorphism seems not be causative for hypospermatogenesis.

Membrane curvature regulates ligand-specific membrane sorting of GPCRs in living cells.

The targeted spatial organization (sorting) of Gprotein-coupled receptors (GPCRs) is essential for their biological function and often takes place in highly curved membrane compartments such as filopodia, endocytic pits, trafficking vesicles or endosome tubules. However, the influence of geometrical membrane curvature on GPCR sorting remains unknown. Here we used fluorescence imaging to establish a quantitative correlation between membrane curvature and sorting of three prototypic class A GPCRs (the neuropeptide Y receptor Y2, the β1 adrenergic receptor and the β2 adrenergic receptor) in living cells. Fitting of a thermodynamic model to the data enabled us to quantify how sorting is mediated by an energetic drive to match receptor shape and membrane curvature. Curvature-dependent sorting was regulated by ligands in a specific manner. We anticipate that this curvature-dependent biomechanical coupling mechanism contributes to the sorting, trafficking and function of transmembrane proteins in general.

GluA1 subunit ubiquitination mediates amyloid-β-induced loss of surface α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors

The accumulation of soluble amyloid-β (Aβ) peptides produces profound neuronal changes in the brain during the pathogenesis of Alzheimer's disease. Excessive levels of Aβ disrupt excitatory synaptic transmission by promoting the removal of synaptic AMPA receptors (AMPARs), dendritic spine loss, and synaptic depression. Recently, activity-dependent ubiquitination of the GluA1 subunit has been shown to regulate the intracellular sorting of AMPARs toward late endosomes for degradation. However, whether this ubiquitin signaling pathway mediates Aβ-induced loss of surface AMPARs is unknown. In this study, we demonstrate that acute exposure of cultured neurons to soluble Aβ oligomers induces AMPAR ubiquitination concomitant with the removal of AMPARs from the plasma membrane. Importantly, expression of the GluA1 ubiquitin-deficient mutants inhibited the adverse effects of Aβ on the surface expression of AMPARs in neurons. Furthermore, we revealed the cross-talk between GluA1 ubiquitination and phosphorylation, in particular phosphorylation at Ser-845, which is crucial for AMPAR recycling and is known to be dephosphorylated in the presence of Aβ. Our data showed that the GluA1 ubiquitin-deficient mutant enhances GluA1 phosphorylation on Ser-845. Conversely, the GluA1 S845D phosphomimetic mutant reduced binding with Nedd4-1 and hence the ubiquitination of AMPARs. Importantly, the GluA1 S845D mutant also prevented Aβ-induced removal of surface AMPARs. Taken together, these findings provide the first demonstration of the dynamic cross-modulation of GluA1 ubiquitination and phosphorylation, a process that is perturbed by Aβ, in regulating the membrane sorting decision that ultimately determines the number of AMPARs on the cell surface.

Magnetic sorting of membrane associated IgG for phenotype-based selection of stable antibody producing cells

To establish a simple and widely accessible technique for rapidly selecting high producing Chinese hamster ovary (CHO) cells engineered to express a monoclonal antibody (mAb), we have exploited the transient display of recombinant protein on their cell surface. In combination with magnetic bead-based methods, we demonstrate the ability to select for cells of high productivity in the absence of any metabolic-based selection method. This technique is sufficient to obtain genetically stable engineered CHO cells via a single step of cell subcloning and yields sought-after stable, high IgG producing clonal cell lines. This technique may also be applied to other types of cells as well as polyclonal Ab cell pools.

Disruption of Rab8a and Rab11a causes formation of basolateral microvilli in neonatal enteropathy.

Misplaced formation of microvilli to basolateral domains and intracellular inclusions in enterocytes are pathognomonic features in congenital enteropathy associated with mutation of the apical plasma membrane receptor syntaxin 3 (STX3). Although the demonstrated binding of Myo5b to the Rab8a and Rab11a small GTPases in vitro implicates cytoskeleton-dependent membrane sorting, the mechanisms underlying the microvillar location defect remain unclear. By selective or combinatory disruption of Rab8a and Rab11a membrane traffic in vivo, we demonstrate that transport of distinct cargo to the apical brush border rely on either individual or both Rab regulators, whereas certain basolateral cargos are redundantly transported by both factors. Enterocyte-specific Rab8a and Rab11a double-knockout mouse neonates showed immediate postnatal lethality and more severe enteropathy than single knockouts, with extensive formation of microvilli along basolateral surfaces. Notably, following an inducible Rab11a deletion from neonatal enterocytes, basolateral microvilli were induced within 3 days. These data identify a potentially important and distinct mechanism for a characteristic microvillus defect exhibited by enterocytes of patients with neonatal enteropathy.

Knockdown of Unconventional Myosin ID Expression Induced Morphological Change in Oligodendrocytes.

Myelin is a special multilamellar structure involved in various functions in the nervous system. In the central nervous system, the oligodendrocyte (OL) produces myelin and has a unique morphology. OLs have a dynamic membrane sorting system associated with cytoskeletal organization, which aids in the production of myelin. Recently, it was reported that the assembly and disassembly of actin filaments is crucial for myelination. However, the partner myosin molecule which associates with actin filaments during the myelination process has not yet been identified. One candidate myosin is unconventional myosin ID (Myo1d) which is distributed throughout central nervous system myelin; however, its function is still unclear. We report here that Myo1d is expressed during later stages of OL differentiation, together with myelin proteolipid protein (PLP). In addition, Myo1d is distributed at the leading edge of the myelin-like membrane in cultured OL, colocalizing mainly with actin filaments, 2′,3′-cyclic nucleotide phosphodiesterase and partially with PLP. Myo1d-knockdown with specific siRNA induces significant morphological changes such as the retraction of processes and degeneration of myelin-like membrane, and finally apoptosis. Furthermore, loss of Myo1d by siRNA results in the impairment of intracellular PLP transport. Together, these results suggest that Myo1d may contribute to membrane dynamics either in wrapping or transporting of myelin membrane proteins during formation and maintenance of myelin.

Zeb2 is essential for Schwann cell differentiation, myelination and nerve repair.

Schwann cell development and peripheral nerve myelination require the serial expression of transcriptional activators, such as Sox10, Oct6/Scip/Pou3f1 and Egr2/Krox20. Here we show that also transcriptional repression, mediated by the zinc-finger protein Zeb2, is essential for differentiation and myelination. Mice lacking Zeb2 in Schwann cells develop a severe peripheral neuropathy, caused by failure of axonal sorting and virtual absence of myelin membranes. Zeb2-deficient Schwann cells continuously express repressors of lineage progression. Moreover, negative regulators of maturation, such as Sox2 and Ednrb, emerge as Zeb2 target genes, supporting its function as an 'inhibitor of inhibitors' in myelination control. When Zeb2 is deleted in adult mice, Schwann cells readily dedifferentiate following peripheral nerve injury and become 'repair cells'. However, nerve regeneration and remyelination are both perturbed, demonstrating that Zeb2, although undetectable in adult Schwann cells, has a latent function throughout life.

Dynamic organization of the mitochondrial protein import machinery.

Mitochondria contain elaborate machineries for the import of precursor proteins from the cytosol. The translocase of the outer mitochondrial membrane (TOM) performs the initial import of precursor proteins and transfers the precursors to downstream translocases, including the presequence translocase and the carrier translocase of the inner membrane, the mitochondrial import and assembly machinery of the intermembrane space, and the sorting and assembly machinery of the outer membrane. Although the protein translocases can function as separate entities in vitro, recent studies revealed a close and dynamic cooperation of the protein import machineries to facilitate efficient transfer of precursor proteins in vivo. In addition, protein translocases were found to transiently interact with distinct machineries that function in the respiratory chain or in the maintenance of mitochondrial membrane architecture. Mitochondrial protein import is embedded in a regulatory network that ensures protein biogenesis, membrane dynamics, bioenergetic activity and quality control.

Multiple Roles of VARP in Endosomal Trafficking: Rabs, Retromer Components and R-SNARE VAMP7 Meet on VARP.

VARP (VPS9-ankyrin-repeat protein, also known as ANKRD27) was originally identified as an N-terminal VPS9 (vacuolar protein sorting 9)-domain-containing protein that possesses guanine nucleotide exchange factor (GEF) activity toward small GTPase Rab21 and contains two ankyrin repeat (ANKR) domains in its central region. A number of VARP-interacting molecules have been identified during the past five years, and considerable attention is now being directed to the multiple roles of VARP in endosomal trafficking. More specifically, VARP is now known to interact with three different types of key membrane trafficking regulators, i.e. small GTPase Rabs (Rab32, Rab38 and Rab40C), the retromer complex (a sorting nexin dimer, VPS26, VPS29 and VPS35) and R-SNARE VAMP7. By binding to several of these molecules, VARP regulates endosomal trafficking, which underlies a variety of cellular events, including melanogenic enzyme trafficking to melanosomes, dendrite outgrowth of melanocytes, neurite outgrowth and retromer-mediated endosome-to-plasma membrane sorting of transmembrane proteins.

Computer Simulations on Interactions between Cytoskeleton-Anchored Peptides and Plasma Membranes

Plasma membranes are not isolated lipid bilayers but are closely coupled with the underlying cytoskeleton through binding some transmembrane proteins with the cytoskeleton. Due to the binding of the cytoskeleton, the lateral diffusion of the transmembrane proteins may be greatly restricted or even become immobilized. Using a coarse-grained MARTINI model, we examined the effects of length and the distributing states of the immobilized WALP peptides on the domain formation of a ternary lipid bilayer. We found that, due to the position and tilt restrictions imposed on the peptides, the effect of hydrophobic mismatch between the peptides and membranes has a significant influence on both the domain formation of membrane and peptide sorting in the membrane.

Spontaneous Tubulation in Giant Vesicles Induced by GM1 or PEG Adsorption

Nanotubes are ubiquitous in cells. Examples for tubular structures are provided not only by organelles such as the Golgi body and the endoplasmic reticulum but also by intercellular connections between different cells. Their geometry and large area-to-volume ratio make them an excellent natural tool for membrane storage, sorting and transport in cells. Such nanotubes can also be formed from synthetic lipid bilayers. Here, we will discuss two systems based on giant vesicles, within which lipid nanotubes are spontaneously generated and stabilized by spontaneous curvature. In the first case, the membrane has asymmetric composition set by the different surface coverage of the ganglioside GM1 on the two leaflets of the membrane. Employing vesicle micromanipulation and electroporation, we could assess the GM1 asymmetry and the associated spontaneous curvature. In the second system, the two leaflets of the bilayer have the same composition, but the solutions in contact with the inner and outer leaflet differ in the concentration of polyethylene glycol (PEG). We show that the spontaneous curvature is now generated by weak adsorption of PEG onto the membranes. The tube shapes, cylindrical or necklace-like, and their diameters can be varied by the membrane composition and bending rigidity. We demonstrate this variation by employing membranes with liquid-ordered and liquid-disordered compositions. This work was supported by the Partner Group Program of the Max Planck Society and the Chinese Academy of Sciences and is part of the MaxSynBio consortium, which is jointly funded by the Federal Ministry of Education and Research of Germany and the Max Planck Society.

HIV-1 Nef hijacks clathrin coats by stabilizing AP-1:Arf1 polygons.

The lentiviruses HIV and simian immunodeficiency virus (SIV) subvert intracellular membrane traffic as part of their replication cycle. The lentiviral Nef protein helps viruses evade innate and adaptive immune defenses by hijacking the adaptor protein 1 (AP-1) and AP-2 clathrin adaptors. We found that HIV-1 Nef and the guanosine triphosphatase Arf1 induced trimerization and activation of AP-1. Here we report the cryo-electron microscopy structures of the Nef- and Arf1-bound AP-1 trimer in the active and inactive states. A central nucleus of three Arf1 molecules organizes the trimers. We combined the open trimer with a known dimer structure and thus predicted a hexagonal assembly with inner and outer faces that bind the membranes and clathrin, respectively. Hexagons were directly visualized and the model validated by reconstituting clathrin cage assembly. Arf1 and Nef thus play interconnected roles in allosteric activation, cargo recruitment, and coat assembly, revealing an unexpectedly intricate organization of the inner AP-1 layer of the clathrin coat.

Golgi fragmentation in Alzheimer's disease.

The Golgi apparatus is an essential cellular organelle for post-translational modifications, sorting, and trafficking of membrane and secretory proteins. Proper functionality of the Golgi requires the formation of its unique cisternal-stacking morphology. The Golgi structure is disrupted in a variety of neurodegenerative diseases, suggesting a common mechanism and contribution of Golgi defects in neurodegenerative disorders. A recent study on Alzheimer's disease (AD) revealed that phosphorylation of the Golgi stacking protein GRASP65 disrupts its function in Golgi structure formation, resulting in Golgi fragmentation. Inhibiting GRASP65 phosphorylation restores the Golgi morphology from Aβ-induced fragmentation and reduces Aβ production. Perturbing Golgi structure and function in neurons may directly impact trafficking, processing, and sorting of a variety of proteins essential for synaptic and dendritic integrity. Therefore, Golgi defects may ultimately promote the development of AD. In the current review, we focus on the cellular impact of impaired Golgi morphology and its potential relationship to AD disease development.

P.7.d.008 The impact of sleep on inattention, impulsiveness and cognitive failures

In the erythrocytes, malaria parasite entry and infection is mediated through complex membrane sorting and signaling processes. We investigated the effects of single-locus and multilocus interactions to test the hypothesis that the members of the GPCR family genes, adenosine A2a receptor (ADORA2A) and G-protein coupled receptor kinase5 (GRK5), may contribute to the pathogenesis of malaria caused by Plasmodium falciparum (Pf) independently or through complex interactions. In a case–control study of adults, individuals affected by Pf malaria (complicated n = 168; uncomplicated n = 282) and healthy controls (n = 450) were tested for their association to four known SNPs in GRK5 (rs2230345, rs2275036, rs4752307 and rs11198918) and two in ADORA2A (rs9624472 and rs5751876) genes with malaria susceptibility, using techniques of polymerase chain reaction-restriction fragment length polymorphisms and direct DNA sequencing. Single-locus analysis showed significant association of 2 SNPs; rs5751876 (OR = 3.2(2.0–5.2); p = 0.0006) of ADORA2A and rs2230345 (OR = 0.3(0.2–0.5); p = 0.0006) of GRK5 with malaria. The mean of the serum creatinine levels were significantly higher in patients with variant GG (p = 0.006) of rs9624472 in ADORA2A gene compared to AA and AG genotypes in complicated Pf malaria cases, with the G allele also showing increased risk for malaria (OR = 1.3(1.1–1.6); p = 0.017). Analyses of predicted haplotypes of the two ADORA2A and the four GRK5 SNPs have identified the haplotypes that conferred risk as well as resistance to malaria with statistical significance. Molecular docking analysis of evolutionary rs2230345 SNP indicated a stable activity of GRK5 for the mutant allele compared to the wild type. Further, generalized multifactor dimensionality reduction to test the contribution of individual effects of the six polymorphisms and higher-order interactions to risk of symptoms/clinical complications of malaria suggested a best six-locus model showing statistical significance. The study provides evidence for the role of ADORA2A and GRK5 that might influence the etiology of malaria infection.