Structure-based Functional Analysis Reveals a Role for the SM Protein Sly1p in Retrograde Transport to the Endoplasmic Reticulum

Sec1p/Munc18 (SM) proteins are essential for membrane fusion events in eukaryotic cells. Here we describe a systematic, structure-based mutational analysis of the yeast SM protein Sly1p, which was previously shown to function in anterograde endoplasmic reticulum (ER)-to-Golgi and intra-Golgi protein transport. Five new temperature-sensitive (ts) mutants, each carrying a single amino acid substitution in Sly1p, were identified. Unexpectedly, not all of the ts mutants exhibited striking anterograde ER-to-Golgi transport defects. For example, in cells of the novel sly1-5 mutant, transport of newly synthesized lysosomal and secreted proteins was still efficient, but the ER-resident Kar2p/BiP was missorted to the outside of the cell, and two proteins, Sed5p and Rer1p, which normally shuttle between the Golgi and the ER, failed to relocate to the ER. We also discovered that in vivo, Sly1p was associated with a SNARE complex formed on the ER, and that in vitro, the SM protein directly interacted with the ER-localized nonsyntaxin SNAREs Use1p/Slt1p and Sec20p. Furthermore, several conditional mutants defective in Golgi-to-ER transport were synthetically lethal with sly1-5. Together, these results indicate a previously unrecognized function of Sly1p in retrograde transport to the endoplasmic reticulum.

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Mutational Analysis of Influenza A Virus Nucleoprotein: Identification of Mutations That Affect RNA Replication

Journal of Virology ◽

10.1128/jvi.73.2.1186-1194.1999 ◽

1999 ◽

Vol 73 (2) ◽

pp. 1186-1194 ◽

Cited By ~ 57

Author(s):

Ignacio Mena ◽

Enrique Jambrina ◽

Carmen Albo ◽

Beatriz Perales ◽

Juan Ortín ◽

...

Keyword(s):

Influenza A Virus ◽

Influenza A ◽

Rna Binding ◽

Mutational Analysis ◽

Intracellular Localization ◽

Single Amino Acid ◽

Temperature Sensitive ◽

Virus Mutant

ABSTRACT The influenza A virus nucleoprotein (NP) is a multifunctional polypeptide which plays a pivotal role in virus replication. To get information on the domains and specific residues involved in the different NP activities, we describe here the preparation and characterization of 20 influenza A virus mutant NPs. The mutations, mostly single-amino-acid substitutions, were introduced in a cDNA copy of the A/Victoria/3/75 NP gene and, in most cases, affected residues located in regions that were highly conserved across the NPs of influenza A, B, and C viruses. The mutant NPs were characterized (i) in vivo (cell culture) by analyzing their intracellular localization and their functionality in replication, transcription, and expression of model RNA templates; and (ii) in vitro by analyzing their RNA-binding and sedimentation properties. The results obtained allowed us to identify both a mutant protein that accumulated in the cytoplasm and mutations that altered the functionality and/or the oligomerization state of the NP polypeptide. Among the mutations that reduced the NP capability to express chloramphenicol acetyltransferase protein from a model viral RNA (vRNA) template, some displayed a temperature-sensitive phenotype. Interestingly, four mutant NPs, which showed a reduced functionality in synthesizing cRNA molecules from a vRNA template, were fully competent to reconstitute complementary ribonucleoproteins (cRNPs) capable of synthesizing vRNAs, which in turn yielded mRNA molecules. Based on the phenotype of these mutants and on previously published observations, it is proposed that these mutant NPs have a reduced capability to interact with the polymerase complex and that this NP-polymerase interaction is responsible for making vRNPs switch from mRNA to cRNA synthesis.

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rsly1 Binding to Syntaxin 5 Is Required for Endoplasmic Reticulum-to-Golgi Transport but Does Not Promote SNARE Motif Accessibility

Molecular Biology of the Cell ◽

10.1091/mbc.e03-07-0535 ◽

2004 ◽

Vol 15 (1) ◽

pp. 162-175 ◽

Cited By ~ 30

Author(s):

Antionette L. Williams ◽

Sebastian Ehm ◽

Noëlle C. Jacobson ◽

Dalu Xu ◽

Jesse C. Hay

Keyword(s):

Endoplasmic Reticulum ◽

Complex Formation ◽

Protein Function ◽

Snare Complex ◽

Golgi Transport ◽

Snare Motif ◽

Sm Protein ◽

Snare Complex Formation

Although some of the principles of N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) function are well understood, remarkably little detail is known about sec1/munc18 (SM) protein function and its relationship to SNAREs. Popular models of SM protein function hold that these proteins promote or maintain an open and/or monomeric pool of syntaxin molecules available for SNARE complex formation. To address the functional relationship of the mammalian endoplasmic reticulum/Golgi SM protein rsly1 and its SNARE binding partner syntaxin 5, we produced a conformation-specific monoclonal antibody that binds only the available, but not the cis-SNARE–complexed nor intramolecularly closed form of syntaxin 5. Immunostaining experiments demonstrated that syntaxin 5 SNARE motif availability is nonuniformly distributed and focally regulated. In vitro endoplasmic reticulum-to-Golgi transport assays revealed that rsly1 was acutely required for transport, and that binding to syntaxin 5 was absolutely required for its function. Finally, manipulation of rsly1–syntaxin 5 interactions in vivo revealed that they had remarkably little impact on the pool of available syntaxin 5 SNARE motif. Our results argue that although rsly1 does not seem to regulate the availability of syntaxin 5, its function is intimately associated with syntaxin binding, perhaps promoting a later step in SNARE complex formation or function.

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Important Role for Phylogenetically Invariant PP2Acα Active Site and C-Terminal Residues Revealed by Mutational Analysis in Saccharomyces cerevisiae

Genetics ◽

10.1093/genetics/156.1.21 ◽

2000 ◽

Vol 156 (1) ◽

pp. 21-29 ◽

Cited By ~ 1

Author(s):

David R H Evans ◽

Brian A Hemmings

Keyword(s):

Protein Interactions ◽

Active Site ◽

Protein Function ◽

Mutational Analysis ◽

Yeast Cells ◽

Temperature Sensitive ◽

Active Site Residues ◽

Catalytic Function

Abstract PP2A is a central regulator of eukaryotic signal transduction. The human catalytic subunit PP2Acα functionally replaces the endogenous yeast enzyme, Pph22p, indicating a conservation of function in vivo. Therefore, yeast cells were employed to explore the role of invariant PP2Ac residues. The PP2Acα Y127N substitution abolished essential PP2Ac function in vivo and impaired catalysis severely in vitro, consistent with the prediction from structural studies that Tyr-127 mediates substrate binding and its side chain interacts with the key active site residues His-118 and Asp-88. The V159E substitution similarly impaired PP2Acα catalysis profoundly and may cause global disruption of the active site. Two conditional mutations in the yeast Pph22p protein, F232S and P240H, were found to cause temperature-sensitive impairment of PP2Ac catalytic function in vitro. Thus, the mitotic and cell lysis defects conferred by these mutations result from a loss of PP2Ac enzyme activity. Substitution of the PP2Acα C-terminal Tyr-307 residue by phenylalanine impaired protein function, whereas the Y307D and T304D substitutions abolished essential function in vivo. Nevertheless, Y307D did not reduce PP2Acα catalytic activity significantly in vitro, consistent with an important role for the C terminus in mediating essential protein-protein interactions. Our results identify key residues important for PP2Ac function and characterize new reagents for the study of PP2A in vivo.

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In-silico structural, functional and immunogenic characterization of Taenia solium TS14 protein

Indian Journal of Animal Research ◽

10.18805/ijar.b-3313 ◽

2017 ◽

Author(s):

Chitra Joshi ◽

Siddharth Gautam

Keyword(s):

Amino Acid ◽

In Silico ◽

Mutational Analysis ◽

Solvent Accessibility ◽

Taenia Solium ◽

Secretory Protein ◽

Single Amino Acid

TS14, a Cysticercosis cellulosae derived protein, has been exploited for immunodiagnosis of cysticercosis in humans and pigs. However, the information on structure, function, stability and immunogenicity of TS14 derived from different isolates is primarily lacking. The present study deals with in-silico characterization of six TS14 isolates. High thermostability and an isoelectric point of 9.41 were recorded. Based on N-terminal amino acid residues, high resistance to intracellular proteases with extended in-vivo and in-vitro half-lives was predicted. TS14 is foreseen as a secretory protein with a signal peptide and an extracellular localization. Structural analysis of TS14 exhibited the dominance of helices in the secondary structure (92% coverage) with majority of residues showing high and medium solvent accessibility. High lysine content and presence of multiple nucleotide binding sites in TS14 suggests interaction with RNA/DNA and a role in their metabolism. Immunogenic profiling predicted presence of four distinct B-cell epitopes. Mutational analysis based on the single amino acid substitutions among six TS14 isolates demonstrated minor variations in structural stability; however, all the substitutions were well tolerated. Moreover, all the isolates revealed almost identical immunogenic profile with an equivocal potential to elicit the antibody-mediated immune response.

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Binding of UNC-18 to the N-terminus of syntaxin is essential for neurotransmission in Caenorhabditis elegans

Biochemical Journal ◽

10.1042/bj20081956 ◽

2009 ◽

Vol 418 (1) ◽

pp. 73-80 ◽

Cited By ~ 43

Author(s):

James R. Johnson ◽

Pawel Ferdek ◽

Lu-Yun Lian ◽

Jeff W. Barclay ◽

Robert D. Burgoyne ◽

...

Keyword(s):

Caenorhabditis Elegans ◽

Membrane Fusion ◽

Snare Complex ◽

Binding Modes ◽

Sm Proteins ◽

Closed Conformation ◽

Physiological Relevance ◽

N Terminus

SNAREs (soluble N-ethylmaleimide-sensitive fusion protein-attachment protein receptors) are widely accepted to drive all intracellular membrane fusion events. SM (Sec1/Munc18-like) proteins bind to SNAREs and this interaction may underlie their ubiquitous requirement for efficient membrane fusion. SM proteins bind to SNAREs in at least three modes: (i) to a closed conformation of syntaxin; (ii) to the syntaxin N-terminus; and (iii) to the assembled SNARE complex. Munc18-1 exhibits all three binding modes and recent in vitro reconstitution assays suggest that its interaction with the syntaxin N-terminus is essential for neuronal SNARE complex binding and efficient membrane fusion. To investigate the physiological relevance of these binding modes, we studied the UNC-18/UNC-64 SM/SNARE pair, which is essential for neuronal exocytosis in Caenorhabditis elegans. Mutations in the N-terminus of UNC-64 strongly inhibited binding to UNC-18, as did mutations targeting closed conformation binding. Complementary mutations in UNC-18 designed to selectively impair binding to either closed syntaxin or its N-terminus produced a similarly strong inhibition of UNC-64 binding. Therefore high-affinity UNC18/UNC-64 interaction in vitro involves both binding modes. To determine the physiological relevance of each mode, unc-18-null mutant worms were transformed with wild-type or mutant unc-18 constructs. The UNC-18(R39C) construct, that is defective in closed syntaxin binding, fully rescued the locomotion defects of the unc-18 mutant. In contrast, the UNC-18(F113R) construct, that is defective in binding to the N-terminus of UNC-64, provided no rescue. These results suggest that binding of UNC-18 to closed syntaxin is dispensable for membrane fusion, whereas interaction with the syntaxin N-terminus is essential for neuronal exocytosis in vivo.

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SM protein Munc18-2 facilitates transition of Syntaxin 11-mediated lipid mixing to complete fusion for T-lymphocyte cytotoxicity

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1617981114 ◽

2017 ◽

Vol 114 (11) ◽

pp. E2176-E2185 ◽

Cited By ~ 20

Author(s):

Waldo A. Spessott ◽

Maria L. Sanmillan ◽

Margaret E. McCormick ◽

Vineet V. Kulkarni ◽

Claudio G. Giraudo

Keyword(s):

Transmembrane Domain ◽

Snare Complex ◽

Complete Fusion ◽

Sm Proteins ◽

Granule Exocytosis ◽

Cytolytic Granule ◽

Syntaxin 11 ◽

Sm Protein ◽

Lytic Granule

The atypical lipid-anchored Syntaxin 11 (STX11) and its binding partner, the Sec/Munc (SM) protein Munc18-2, facilitate cytolytic granule release by cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells. Patients carrying mutations in these genes develop familial hemophagocytic lymphohistiocytosis, a primary immunodeficiency characterized by impaired lytic granule exocytosis. However, whether a SNARE such as STX11, which lacks a transmembrane domain, can support membrane fusion in vivo is uncertain, as is the precise role of Munc18-2 during lytic granule exocytosis. Here, using a reconstituted “flipped” cell–cell fusion assay, we show that lipid-anchored STX11 and its cognate SNARE proteins mainly support exchange of lipids but not cytoplasmic content between cells, resembling hemifusion. Strikingly, complete fusion is stimulated by addition of wild-type Munc18-2 to the assay, but not of Munc18-2 mutants with abnormal STX11 binding. Our data reveal that Munc18-2 is not just a chaperone of STX11 but also directly contributes to complete membrane merging by promoting SNARE complex assembly. These results further support the concept that SM proteins in general are part of the core fusion machinery. This fusion mechanism likely contributes to other cell-type–specific exocytic processes such as platelet secretion.

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p62cdc23 of Saccharomyces cerevisiae: a nuclear tetratricopeptide repeat protein with two mutable domains.

Molecular and Cellular Biology ◽

10.1128/mcb.13.2.1212 ◽

1993 ◽

Vol 13 (2) ◽

pp. 1212-1221 ◽

Cited By ~ 43

Author(s):

R S Sikorski ◽

W A Michaud ◽

P Hieter

Keyword(s):

Saccharomyces Cerevisiae ◽

Cell Cycle Progression ◽

Single Amino Acid ◽

Temperature Sensitive ◽

Cycle Progression ◽

Tetratricopeptide Repeats ◽

Tetratricopeptide Repeat Protein ◽

Cdc Genes

CDC23 is required in Saccharomyces cerevisiae for cell cycle progression through the G2/M transition. The CDC23 gene product contains tandem, imperfect repeats, termed tetratricopeptide repeats, (TPR) units common to a protein family that includes several other nuclear division CDC genes. In this report we have used mutagenesis to probe the functional significance of the TPR units within CDC23. Analysis of truncated derivatives indicates that the TPR block of CDC23 is necessary for the function or stability of the polypeptide. In-frame deletion of a single TPR unit within the repeat block proved sufficient to inactivate CDC23 in vivo, though this allele could rescue the temperature-sensitive defect of a cdc23 point mutant by intragenic complementation. By both in vitro and in vivo mutagenesis techniques, 17 thermolabile cdc23 alleles were produced and examined. Fourteen alleles contained single amino acid changes that were found to cluster within two distinct mutable domains, one of which encompasses the most canonical TPR unit found in CDC23. In addition, we have characterized CDC23 as a 62-kDa protein (p62cdc23) that is localized to the yeast nucleus. Our mutagenesis results suggest that TPR blocks form an essential domain within members of the TPR family.

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Golgi SM protein Sly1 promotes productive trans-SNARE complex assembly through multiple mechanisms

10.1101/2020.01.16.909630 ◽

2020 ◽

Author(s):

M. Duan ◽

G. Gao ◽

D.K. Banfield ◽

A.J. Merz

Keyword(s):

Snare Complex ◽

Complex Assembly ◽

Present Evidence ◽

Closed Conformation ◽

Close Range ◽

Preferential Nucleation ◽

Regulatory Loop ◽

Sm Protein

SUMMARYSNARE chaperones of the Sec1/mammalian Unc-18 (SM) family have critical roles in SNARE-mediated membrane fusion. Using SNARE and Sly1 mutants, and a new in vitro assay of fusion, we separate and assess proposed mechanisms through which Sly1 augments fusion: (i) opening the closed conformation of the Qa-SNARE Sed5; (ii) close-range tethering of vesicles to target organelles, mediated by the Sly1-specific regulatory loop; and (iii) preferential nucleation of productive trans-SNARE complexes. We show that all three mechanisms are important and operate in parallel, and we present evidence that close-range tethering is particularly important for trans-complex assembly when cis-SNARE assembly is a competing process. In addition, the autoinhibitory N-terminal Habc domain of Sed5 has at least two positive activities: the Habc domain is needed for correct Sed5 localization, and it directly promotes Sly1-dependent fusion. Remarkably, “split Sed5,” with the Habc domain present only as a soluble fragment, is functional both in vitro and in vivo.

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SM proteins Sly1 and Vps33 co-assemble with Sec17 and SNARE complexes to oppose SNARE disassembly by Sec18

eLife ◽

10.7554/elife.02272 ◽

2014 ◽

Vol 3 ◽

Cited By ~ 50

Author(s):

Braden T Lobingier ◽

Daniel P Nickerson ◽

Sheng-Ying Lo ◽

Alexey J Merz

Keyword(s):

Snare Complex ◽

Wild Type ◽

Sm Proteins ◽

Working Model ◽

Conformational Rearrangements ◽

Kinetic Proofreading

Secretory and endolysosomal fusion events are driven by SNAREs and cofactors, including Sec17/α-SNAP, Sec18/NSF, and Sec1/Munc18 (SM) proteins. SMs are essential for fusion in vivo, but the basis of this requirement is enigmatic. We now report that, in addition to their established roles as fusion accelerators, SM proteins Sly1 and Vps33 directly shield SNARE complexes from Sec17- and Sec18-mediated disassembly. In vivo, wild-type Sly1 and Vps33 function are required to withstand overproduction of Sec17. In vitro, Sly1 and Vps33 impede SNARE complex disassembly by Sec18 and ATP. Unexpectedly, Sec17 directly promotes selective loading of Sly1 and Vps33 onto cognate SNARE complexes. A large thermodynamic barrier limits SM binding, implying that significant conformational rearrangements are involved. In a working model, Sec17 and SMs accelerate fusion mediated by cognate SNARE complexes and protect them from NSF-mediated disassembly, while mis-assembled or non-cognate SNARE complexes are eliminated through kinetic proofreading by Sec18.

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Dissociation of Coatomer from Membranes Is Required for Brefeldin A–induced Transfer of Golgi Enzymes to the Endoplasmic Reticulum

The Journal of Cell Biology ◽

10.1083/jcb.137.2.319 ◽

1997 ◽

Vol 137 (2) ◽

pp. 319-333 ◽

Cited By ~ 60

Author(s):

Jochen Scheel ◽

Rainer Pepperkok ◽

Martin Lowe ◽

Gareth Griffiths ◽

Thomas E. Kreis

Keyword(s):

Endoplasmic Reticulum ◽

Membrane Transport ◽

Golgi Complex ◽

Mammalian Cells ◽

Brefeldin A ◽

Retrograde Transport ◽

Marker Proteins ◽

Kdel Receptor

Addition of brefeldin A (BFA) to mammalian cells rapidly results in the removal of coatomer from membranes and subsequent delivery of Golgi enzymes to the endoplasmic reticulum (ER). Microinjected anti-EAGE (intact IgG or Fab-fragments), antibodies against the “EAGE”-peptide of β-COP, inhibit BFA-induced redistribution of β-COP in vivo and block transfer of resident proteins of the Golgi complex to the ER; tubulo-vesicular clusters accumulate and Golgi membrane proteins concentrate in cytoplasmic patches containing β-COP. These patches are devoid of marker proteins of the ER, the intermediate compartment (IC), and do not contain KDEL receptor. Interestingly, relocation of KDEL receptor to the IC, where it colocalizes with ERGIC53 and ts-O45-G, is not inhibited under these conditions. While no stacked Golgi cisternae remain in these injected cells, reassembly of stacks of Golgi cisternae following BFA wash-out is inhibited to only ∼50%. Mono- or divalent anti-EAGE stabilize binding of coatomer to membranes in vitro, at least as efficiently as GTPγS. Taken together these results suggest that enhanced binding of coatomer to membranes completely inhibits the BFA-induced retrograde transport of Golgi resident proteins to the ER, probably by inhibiting fusion of Golgi with ER membranes, but does not interfere with the disassembly of the stacked Golgi cisternae and recycling of KDEL receptor to the IC. These results confirm our previous results suggesting that COPI is involved in anterograde membrane transport from the ER/IC to the Golgi complex (Pepperkok et al., 1993), and corroborate that COPI regulates retrograde membrane transport between the Golgi complex and ER in mammalian cells.

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