scholarly journals Analysis of the SNARE Stx8 recycling reveals that the retromer-sorting motif has undergone evolutionary divergence

PLoS Genetics ◽  
2021 ◽  
Vol 17 (3) ◽  
pp. e1009463
Author(s):  
Francisco Yanguas ◽  
M.-Henar Valdivieso
Keyword(s):  
Computer Assisted ◽  
Evolutionary Divergence ◽  
Aliphatic Chain ◽  
Hydrophobic Residue ◽  
Alanine Scanning Mutagenesis ◽  
Sorting Nexins ◽  
Experimental Approaches ◽  
Sorting Motif ◽  
Golgi Network ◽  
Unstructured Protein

Fsv1/Stx8 is a Schizosaccharomyces pombe protein similar to mammalian syntaxin 8. stx8Δ cells are sensitive to salts, and the prevacuolar endosome (PVE) is altered in stx8Δ cells. These defects depend on the SNARE domain, data that confirm the conserved function of syntaxin8 and Stx8 in vesicle fusion at the PVE. Stx8 localizes at the trans-Golgi network (TGN) and the prevacuolar endosome (PVE), and its recycling depends on the retromer component Vps35, and on the sorting nexins Vps5, Vps17, and Snx3. Several experimental approaches demonstrate that Stx8 is a cargo of the Snx3-retromer. Using extensive truncation and alanine scanning mutagenesis, we identified the Stx8 sorting signal. This signal is an IEMeaM sequence that is located in an unstructured protein region, must be distant from the transmembrane (TM) helix, and where the 133I, 134E, 135M, and 138M residues are all essential for recycling. This sorting motif is different from those described for most retromer cargoes, which include aromatic residues, and resembles the sorting motif of mammalian polycystin-2 (PC2). Comparison of Stx8 and PC2 motifs leads to an IEMxx(I/M) consensus. Computer-assisted screening for this and for a loose Ψ(E/D)ΨXXΨ motif (where Ψ is a hydrophobic residue with large aliphatic chain) shows that syntaxin 8 and PC2 homologues from other organisms bear variation of this motif. The phylogeny of the Stx8 sorting motifs from the Schizosaccharomyces species shows that their divergence is similar to that of the genus, showing that they have undergone evolutionary divergence. A preliminary analysis of the motifs in syntaxin 8 and PC2 sequences from various organisms suggests that they might have also undergone evolutionary divergence, what suggests that the presence of almost-identical motifs in Stx8 and PC2 might be a case of convergent evolution.

scholarly journals A Novel PDZ Domain Interaction Mediates the Binding between Human Papillomavirus 16 L2 and Sorting Nexin 27 and Modulates Virion Trafficking

2015 ◽  
Vol 89 (20) ◽  
pp. 10145-10155 ◽  
Author(s):  
David Pim ◽  
Justyna Broniarczyk ◽  
Martina Bergant ◽  
Martin P. Playford ◽  
Lawrence Banks
Keyword(s):  
Pdz Domain ◽  
Endosomal Trafficking ◽  
Sorting Nexins ◽  
Human Papillomavirus 16 ◽  
Sorting Nexin ◽  
Papillomavirus Infection ◽  
Infection Dynamics ◽  
And Function ◽  
Golgi Network ◽  
Interfering Rna

ABSTRACTPrevious studies have demonstrated an interaction between sorting nexin 17 and the L2 capsid proteins from a variety of papillomavirus types. This interaction is required for late endosomal trafficking of the L2 protein and entry of the L2/DNA complex into the nucleus during infection. Here we show an interaction between papillomavirus L2 proteins and the related PX-FERM family member sorting nexin 27 (SNX27), which is mediated in part by a novel interaction between the PDZ domain of SNX27 and sequences in a central portion of L2. The interaction is direct and, unlike that with SNX17, is variable in strength depending on the papillomavirus type. We show that small interfering RNA (siRNA)-mediated knockdown of SNX27 alone leads to a marginal reduction in the efficiency of viral infection but that double knockdown of both sorting nexins results in a striking reduction in infection, greater than that observed for the knockdown of either sorting nexin alone. These results suggest that the HPV L2 proteins can interact through distinct mechanisms with multiple components of the cellular cargo-sorting machinery.IMPORTANCEThe trafficking of papillomaviruses to the host cell nucleus during their natural infectious life cycle is an incompletely understood process. Studies have suggested that the virus minor capsid protein L2 can interact with the endosomal recycling pathway, in part by association with sorting nexin 17, to ensure that virus DNA bound to L2 is recycled through the trans-Golgi network rather than back to the plasma membrane. In this study, we characterize the interaction between L2 and a second sorting nexin, SNX27, which is also part of the retromer complex. The study furthers our understanding of papillomavirus infection dynamics and provides potential tools for the further dissection of endosomal structure and function.

scholarly journals Sequence-dependent cargo recognition by SNX-BARs mediates retromer-independent transport of CI-MPR

2017 ◽  
Vol 216 (11) ◽  
pp. 3695-3712 ◽  
Author(s):  
Boris Simonetti ◽  
Chris M. Danson ◽  
Kate J. Heesom ◽  
Peter J. Cullen
Keyword(s):  
Transmembrane Proteins ◽  
Endosomal Sorting ◽  
Sorting Nexin ◽  
Sequence Dependent ◽  
Endosomal Recycling ◽  
Cytosolic Domains ◽  
Sorting Motif ◽  
Golgi Network ◽  
Coupling Sequence

Endosomal recycling of transmembrane proteins requires sequence-dependent recognition of motifs present within their intracellular cytosolic domains. In this study, we have reexamined the role of retromer in the sequence-dependent endosome-to–trans-Golgi network (TGN) transport of the cation-independent mannose 6-phosphate receptor (CI-MPR). Although the knockdown or knockout of retromer does not perturb CI-MPR transport, the targeting of the retromer-linked sorting nexin (SNX)–Bin, Amphiphysin, and Rvs (BAR) proteins leads to a pronounced defect in CI-MPR endosome-to-TGN transport. The retromer-linked SNX-BAR proteins comprise heterodimeric combinations of SNX1 or SNX2 with SNX5 or SNX6 and serve to regulate the biogenesis of tubular endosomal sorting profiles. We establish that SNX5 and SNX6 associate with the CI-MPR through recognition of a specific WLM endosome-to-TGN sorting motif. From validating the CI-MPR dependency of SNX1/2–SNX5/6 tubular profile formation, we provide a mechanism for coupling sequence-dependent cargo recognition with the biogenesis of tubular profiles required for endosome-to-TGN transport. Therefore, the data presented in this study reappraise retromer’s role in CI-MPR transport.

scholarly journals Directional Transneuronal Infection by Pseudorabies Virus Is Dependent on an Acidic Internalization Motif in the Us9 Cytoplasmic Tail

2000 ◽  
Vol 74 (10) ◽  
pp. 4549-4561 ◽  
Author(s):  
Amy D. Brideau ◽  
Marlies G. Eldridge ◽  
Lynn W. Enquist
Keyword(s):  
Plasma Membrane ◽  
Pseudorabies Virus ◽  
Cytoplasmic Tail ◽  
Amino Acid Type ◽  
Alanine Scanning Mutagenesis ◽  
Tyrosine Residues ◽  
Acid Type ◽  
Virion Envelope ◽  
Golgi Network

ABSTRACT The Us9 gene is conserved among most alphaherpesviruses. In pseudorabies virus (PRV), the Us9 protein is a 98-amino-acid, type II membrane protein found in the virion envelope. It localizes to thetrans-Golgi network (TGN) region in infected and transfected cells and is maintained in this compartment by endocytosis from the plasma membrane. Viruses with Us9 deleted have no observable defects in tissue culture yet have reduced virulence and restricted spread to retinorecipient neurons in the rodent brain. In this report, we demonstrate that Us9-promoted transneuronal spread in vivo is dependent on a conserved acidic motif previously shown to be essential for the maintenance of Us9 in the TGN region and recycling from the plasma membrane. Mutant viruses with the acidic motif deleted have an anterograde spread defect indistinguishable from that of Us9 null viruses. Transneuronal spread, however, is not dependent on a dileucine endocytosis motif in the Us9 cytoplasmic tail. Through alanine scanning mutagenesis of the acidic motif, we have identified two conserved tyrosine residues that are essential for Us9-mediated spread as well as two serine residues, comprising putative consensus casein kinase II sites, that modulate the rate of PRV transneuronal spread in vivo.

A Computer-Assisted Systematic Search for Melatonin Derivatives with High Potential as Antioxidants

2018 ◽  
Vol 1 (1) ◽  
pp. 27-58 ◽  
Author(s):  
Miguel Reina ◽  
Romina Castañeda-Arriaga ◽  
Adriana Perez-Gonzalez ◽  
Eduardo Gabriel Guzman-Lopez ◽  
Dun-Xian Tan ◽  
...  
Keyword(s):  
Electron Transfer ◽  
Free Radicals ◽  
Search Space ◽  
Water Soluble ◽  
Computer Assisted ◽  
Reactivity Indices ◽  
Water Soluble Vitamin ◽  
Synthetic Accessibility ◽  
Experimental Approaches ◽  
Selection Of

     A systematic rational search for newly designed melatonin derivatives, was performed using a computer-assisted protocol. A total of 116 derivatives were generated by adding functional groups (i.e., -OH, -NH2, -SH and -COOH) to the melatonin structure. A selection score (SS) was built to sample the search space, simultaneously considering ADME (absorption, distribution, metabolism, excretion) properties, toxicity and manufacturability (i.e., synthetic accessibility). The search characterized the whole set of designed melatonin derivatives and allowed the selection of a reduced subset of 20 melatonin derivatives that are expected to be the most promising, regarding drug-like behavior. For this subset, several reactivity indices were estimated, as well as their pKa values. According to the gathered data, 5 melatonin derivatives have been identified as the most likely candidates to act as chemical antioxidant (directly scavenging free radicals, by electron transfer and/or H transfer). All of them are predicted to be better for that purpose than melatonin itself or trolox (water soluble vitamin E analog). The findings from this work are expected to motivate further investigations on these molecules, using both theoretical and experimental approaches.

scholarly journals Env7p Associates with the Golgin Protein Imh1 at the trans-Golgi Network in Candida albicans

mSphere ◽  
2016 ◽  
Vol 1 (4) ◽  
Author(s):  
Kongara Hanumantha Rao ◽  
Swagata Ghosh ◽  
Asis Datta
Keyword(s):  
Candida Albicans ◽  
Protein Kinase ◽  
Functional Characterization ◽  
Evolutionary Divergence ◽  
Content Type ◽  
Membrane Asymmetry ◽  
Specific Localization ◽  
Golgi Network

ABSTRACT A multitier regulation exists at the trans-Golgi network in all higher organisms. We report a palmitoylated protein kinase, Env7, that functions at the TGN interface by interacting with two more TGN-resident proteins, namely, Imh1 and Arl1. Palmitoylation seems to be important for the specific localization. This study focuses on the involvement of a ubiquitous protein kinase, whose substrates had not yet been reported from any organism, as an upstream signaling component that modulates the activity of the Imh1-Arl1 complex crucial for maintaining membrane asymmetry. Virulence is significantly diminished in an Env7 mutant. The functioning of this protein in C. albicans seems to be quite different from its nearest homologue in S. cerervisiae, which reflects the evolutionary divergence between these two organisms. Vesicular dynamics is one of the very important aspects of cellular physiology, an imbalance of which leads to the disorders or diseases in higher eukaryotes. We report the functional characterization of a palmitoylated protein kinase from Candida albicans whose homologue in Saccharomyces cerevisiae has been reported to be involved in negative regulation of membrane fusion and was named Env7. However, the downstream target of this protein remains to be identified. Env7 in C. albicans (CaEnv7) could be isolated from the membrane fraction and localized to vesicular structures associated with the Golgi apparatus. Our work reports Env7 in C. albicans as a new player involved in maintaining the functional dynamics at the trans-Golgi network (TGN) by interacting with two other TGN-resident proteins, namely, Imh1p and Arl1p. Direct interaction could be detected between Env7p and the golgin protein Imh1p. Env7 is itself phosphorylated (Env7p) and phosphorylates Imh1 in vivo. An interaction between Env7 and Imh1 is required for the targeted localization of Imh1. CaEnv7 has a putative palmitoylation site toward both N and C termini. An N-terminal palmitoylation-defective strain retains its ability to phosphorylate Imh1 in vitro. An ENV7 homozygous mutant showed compromised filamentation in solid media and attenuated virulence, whereas an overexpressed strain affected cell wall integrity. Thus, Env7 plays a subtle but important role at the level of multitier regulation that exists at the TGN. IMPORTANCE A multitier regulation exists at the trans-Golgi network in all higher organisms. We report a palmitoylated protein kinase, Env7, that functions at the TGN interface by interacting with two more TGN-resident proteins, namely, Imh1 and Arl1. Palmitoylation seems to be important for the specific localization. This study focuses on the involvement of a ubiquitous protein kinase, whose substrates had not yet been reported from any organism, as an upstream signaling component that modulates the activity of the Imh1-Arl1 complex crucial for maintaining membrane asymmetry. Virulence is significantly diminished in an Env7 mutant. The functioning of this protein in C. albicans seems to be quite different from its nearest homologue in S. cerervisiae, which reflects the evolutionary divergence between these two organisms.

The Golgi-targeting sequence of the peripheral membrane protein p230

1999 ◽  
Vol 112 (11) ◽  
pp. 1645-1654 ◽  
Author(s):  
L. Kjer-Nielsen ◽  
C. van Vliet ◽  
R. Erlich ◽  
B.H. Toh ◽  
P.A. Gleeson
Keyword(s):  
Fusion Protein ◽  
Fluorescent Protein ◽  
Brefeldin A ◽  
Deletion Mutants ◽  
Alanine Scanning Mutagenesis ◽  
Binding Characteristics ◽  
Cos Cells ◽  
Trans Golgi Network ◽  
Green Fluorescent ◽  
Golgi Network

Vesicle transport requires the recruitment of cytosolic proteins to specific membrane compartments. We have previously characterised a brefeldin A-sensitive trans-Golgi network-localised protein (p230) that is associated with a population of non-clathrin-coated vesicles. p230 recycles between the cytosol and the cytoplasmic face of buds/vesicles of trans-Golgi network membranes in a G protein-regulated manner. Identifying the mechanism responsible for Golgi targeting of p230 is important for the elucidation of its function. By transfection of COS cells with deletion mutants of p230 we here demonstrate that the C-terminal domain is necessary for targeting to the Golgi. Furthermore, the C-terminal 98 amino acid domain of p230 attached to the green fluorescent protein (GFP-p230-C98aa) was efficiently Golgi-localised in transfected COS cells. Deletion mutants of GFP-p230-C98aa together with alanine scanning mutagenesis identified a minimum stretch of 42 amino acids that is essential for Golgi targeting, suggesting that the conformation of the domain is critical for efficient targeting. In COS cells expressing high levels of GFP-p230-C98aa fusion protein, endogenous p230 was no longer associated with Golgi membranes, suggesting that the GFP fusion protein and endogenous p230 may compete for the same membrane target structures. The Golgi binding of GFP-p230-C98aa is brefeldin A-sensitive and is regulated by G proteins. These studies have identified a minimal sequence responsible for specific targeting of p230 to the Golgi apparatus, which displays similar membrane binding characteristics to wild-type p230.

scholarly journals The tyrosine motifs of Lamp 1 and LAP determine their direct and indirect targetting to lysosomes

2002 ◽  
Vol 115 (1) ◽  
pp. 185-194
Author(s):  
Stefanie Obermüller ◽  
Christina Kiecke ◽  
Kurt von Figura ◽  
Stefan Höning
Keyword(s):  
Plasma Membrane ◽  
Indirect Pathway ◽  
Vitro Binding ◽  
Cytoplasmic Tails ◽  
Sorting Motif ◽  
Lysosomal Membrane Proteins ◽  
Necessary And Sufficient ◽  
Golgi Network ◽  
Mutant Forms

Lamp 1 and lysosomal acid phosphatase (LAP) are lysosomal membrane proteins that harbour a tyrosine-based sorting motif within their short cytoplasmic tails. Lamp 1 is delivered from the trans-Golgi network (TGN) via endosomes directly to lysosomes bypassing the plasma membrane, whereas LAP is indirectly transported to lysosomes and recycles between endosomes and the plasma membrane before being delivered to lysosomes.By analysing truncated forms of LAP and chimeras in which the cytoplasmic tail or part of the cytoplasmic tails of LAP and Lamp 1 were exchanged, we were able to show that the YRHV tyrosine motif of LAP is necessary and sufficient to mediate recycling between endosomes and the plasma membrane. When peptides corresponding to the cytoplasmic tails of LAP and Lamp 1 and chimeric or mutant forms of these tails were assayed for in vitro binding of AP1 and AP2, we found that AP2 bound to LAP- and Lamp-1-derived peptides, whereas AP1 bound only to peptides containing the YQTI tyrosine motif of Lamp 1. Residues +2 and +3 of the tyrosine motif were critical for the differential binding of adaptors. LAP in which these residues (–HV) were substituted for those of Lamp 1 (–TI) was transported directly to lysosomes, whereas a chimera carrying the Lamp 1 tail in which residues +2 and +3 were substituted for those of LAP (–HV) gained the ability to recycle. In conclusion, the residues +2 and +3 of the tyrosine motifs determine the sorting of Lamp 1 and LAP in endosomes, mediating either the direct or the indirect pathway to lysosomes.

scholarly journals The targeting of Lamp1 to lysosomes is dependent on the spacing of its cytoplasmic tail tyrosine sorting motif relative to the membrane.

1996 ◽  
Vol 132 (4) ◽  
pp. 565-576 ◽  
Author(s):  
J Rohrer ◽  
A Schweizer ◽  
D Russell ◽  
S Kornfeld
Keyword(s):  
Amino Acids ◽  
Plasma Membrane ◽  
Amino Acid ◽  
Cytoplasmic Tail ◽  
Kinetic Studies ◽  
Type I ◽  
Trans Golgi Network ◽  
Intracellular Targeting ◽  
Sorting Motif ◽  
Golgi Network

Lamp1 is a type I transmembrane glycoprotein that is localized primarily in lysosomes and late endosomes. Newly synthesized molecules are mostly transported from the trans-Golgi network directly to endosomes and then to lysosomes. A minor pathway involves transport via the plasma membrane. The 11-amino acid cytoplasmic tail of lamp1 contains a tyrosine-based motif that has been previously shown to mediate sorting in the trans-Golgi network and rapid internalization at the plasma membrane. We studied whether this motif also mediates sorting in endosomes. We found that mutant forms of lamp1 in which all the amino acids of the cytoplasmic tail were modified except for the RKR membrane anchor and the YXXI sorting motif still localized to dense lysosomes, indicating that the YXXI motif is sufficient to confer proper intracellular targeting. However, when the spacing of the YXXI motif relative to the membrane was changed by deleting one amino acid or adding five amino acids, lysosomal targeting was almost completely abolished. Kinetic studies showed that these mutants were trapped in a recycling pathway, involving trafficking between the plasma membrane and early endocytic compartments. These findings indicate that the YXXI signal of lamp1 is recognized at several sorting sites, including the trans-Golgi network, the plasma membrane, and the early/sorting endosomes. Small changes in the spacing of this motif relative to the membrane dramatically impair sorting in the early/sorting endosomes but have only a modest effect on internalization at the plasma membrane. The spacing of sorting signals relative to the membrane may prove to be an important determinant in the functioning of these signals.

3-D organization of fibrinogen receptors using computer reconstruction and whole-mount microscopy

1988 ◽  
Vol 46 ◽  
pp. 30-31
Author(s):  
E. T. O'Toole ◽  
R. R. Hantgan ◽  
J. C. Lewis
Keyword(s):  
Whole Blood ◽  
Colloidal Gold ◽  
Blood Platelets ◽  
Cell Contact ◽  
Computer Assisted ◽  
Adherent Cells ◽  
Differential Centrifugation ◽  
Computer Reconstruction ◽  
Sds Page ◽  
Whole Mount

Thrombocytes (TC), the avian equivalent of blood platelets, support hemostasis by aggregating at sites of injury. Studies in our lab suggested that fibrinogen (fib) is a requisite cofactor for TC aggregation but operates by an undefined mechanism. To study the interaction of fib with TC and to identify fib receptors on cells, fib was purified from pigeon plasma, conjugated to colloidal gold and used both to facilitate aggregation and as a receptor probe. Described is the application of computer assisted reconstruction and stereo whole mount microscopy to visualize the 3-D organization of fib receptors at sites of cell contact in TC aggregates and on adherent cells.Pigeon TC were obtained from citrated whole blood by differential centrifugation, washed with Ca++ free Hank's balanced salts containing 0.3% EDTA (pH 6.5) and resuspended in Ca++ free Hank's. Pigeon fib was isolated by precipitation with PEG-1000 and the purity assessed by SDS-PAGE. Fib was conjugated to 25nm colloidal gold by vortexing and the conjugates used as the ligand to identify fib receptors.

Computer Assisted Image Reconstruction of Oligomer Structure

1976 ◽  
Vol 34 ◽  
pp. 472-473
Author(s):  
A.M. Jones ◽  
A. Max Fiskin
Keyword(s):  
Three Dimensional ◽  
Depth Of Field ◽  
Computer Assisted ◽  
Projection Data ◽  
Two Dimensional ◽  
Single Particles ◽  
Dimensional Reconstruction ◽  
Computer Assisted Image ◽  
The Fourier Transform ◽  
Space Approach

If the tilt of a specimen can be varied either by the strategy of observing identical particles orientated randomly or by use of a eucentric goniometer stage, three dimensional reconstruction procedures are available (l). If the specimens, such as small protein aggregates, lack periodicity, direct space methods compete favorably in ease of implementation with reconstruction by the Fourier (transform) space approach (2). Regardless of method, reconstruction is possible because useful specimen thicknesses are always much less than the depth of field in an electron microscope. Thus electron images record the amount of stain in columns of the object normal to the recording plates. For single particles, practical considerations dictate that the specimen be tilted precisely about a single axis. In so doing a reconstructed image is achieved serially from two-dimensional sections which in turn are generated by a series of back-to-front lines of projection data.

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