Distinct regions of the Haloferax volcanii dolichol phosphate-mannose synthase AglD mediate the assembly and subsequent processing of the lipid-linked mannose

2021 ◽  
Author(s):  
Marianna Zaretsky ◽  
Ziqiang Guan ◽  
Raz Zarivach ◽  
Jerry Eichler
Keyword(s):  
Plasma Membrane ◽  
Catalytic Domain ◽  
Transmembrane Domain ◽  
Structural Information ◽  
Pyrococcus Furiosus ◽  
3D Structure ◽  
Haloferax Volcanii ◽  
Subsequent Step ◽  
Membrane Spanning ◽  
In Cells

Haloferax volcanii AglD is currently the only archaeal dolichol phosphate (DolP)-mannose synthase shown to participate in N-glycosylation. However, the relation between AglD and Pyrococcus furiosus PF0058, the only archaeal DolP-mannose synthase for which structural information is presently available, was unclear. In this report, similarities between the PF0058 and AglD catalytic domains were revealed. At the same time, AglD includes a transmembrane domain far longer than that of PF0058 or other DolP-mannose synthases. To determine whether this extension affords AglD functions in addition to generating mannose-charged DolP, a series of Hfx. volcanii strains expressing truncated versions of AglD was generated. Mass spectrometry revealed that a version of AglD comprising the catalytic domain and only two of the six to nine predicted membrane-spanning domains could mediate mannose addition to DolP. However, in cells expressing this or other truncated versions of AglD, mannose was not transferred from the lipid to the protein-bound tetrasaccharide precursor of the N-linked pentasaccharide normally decorating Hfx. volcanii glycoproteins. These results thus point to AglD as contributing to additional aspects of Hfx. volcanii N-glycosylation beyond charging DolP with mannose. Accordingly, the possibility that AglD, possibly in coordination with AglR, translocates DolP-mannose across the plasma membrane is discussed. Layman summary In the archaea Haloferax volcanii , the dolichol phosphate (DolP)-mannose synthase AglD charges the lipid DolP with mannose, which is delivered to a protein-bound tetrasaccharide to generate the pentasaccharide decorating glycoproteins in this organism. Structural studies demonstrated the similarity of AglD to Pyrococcus furiosus PF0058, the only archaeal DolP-mannose synthase with a solved 3D structure. Truncated AglD containing the catalytic domain and only two of the predicted six to nine membrane-spanning regions catalyzed mannose-charging of DolP. Yet, no mannose was delivered to protein-linked tetrasaccharide in cells expressing AglD mutants including only up to five membrane-spanning regions, pointing to a role for the extended C-terminal region in a subsequent step of Hfx. volcanii N-glycosylation, such as DolP-mannose translocation across the plasma membrane.

scholarly journals Subcellular Localization and Topology of the p7 Polypeptide of Hepatitis C Virus

2002 ◽  
Vol 76 (8) ◽  
pp. 3720-3730 ◽  
Author(s):  
Séverine Carrère-Kremer ◽  
Claire Montpellier-Pala ◽  
Laurence Cocquerel ◽  
Czeslaw Wychowski ◽  
François Penin ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Subcellular Localization ◽  
Secretory Pathway ◽  
Transmembrane Domain ◽  
Signal Sequence ◽  
C Terminus ◽  
Membrane Spanning ◽  
Polytopic Membrane Protein

ABSTRACT Although biological and biochemical data have been accumulated on most hepatitis C virus proteins, the structure and function of the 63-amino-acid p7 polypeptide of this virus have never been investigated. In this work, sequence analyses predicted that p7 contains two transmembrane passages connected by a short hydrophilic segment. The C-terminal transmembrane domain of p7 was predicted to function as a signal sequence, which was confirmed experimentally by analyzing the translocation of a reporter glycoprotein fused at its C terminus. The p7 polypeptide was tagged either with the ectodomain of CD4 or with a Myc epitope to study its membrane integration, its subcellular localization, and its topology. Alkaline extraction studies confirmed that p7 is an integral membrane polypeptide. The CD4-p7 chimera was detected by immunofluorescence on the surface of nonpermeabilized cells, indicating that it is exported to the plasma membrane. However, pulse-chase analyses showed that only approximately 20% of endoglycosidase H-resistant CD4-p7 was detected after long chase times, suggesting that a large proportion of p7 stays in an early compartment of the secretory pathway. Finally, by inserting a Myc epitope in several positions of p7 and analyzing the accessibility of this epitope on the plasma membrane of HepG2 cells, we showed that p7 has a double membrane-spanning topology, with both its N and C termini oriented toward the extracellular environment. Altogether, these data indicate that p7 is a polytopic membrane protein that could have a functional role in several compartments of the secretory pathway.

scholarly journals TMEM120A contains a specific coenzyme A-binding site and might not mediate poking- or stretch-induced channel activities in cells

2021 ◽  
Author(s):  
Yao Rong ◽  
Jinghui Jiang ◽  
Yiwei Gao ◽  
Jianli Guo ◽  
Danfeng Song ◽  
...  
Keyword(s):  
Coenzyme A ◽  
Transmembrane Domain ◽  
Transmembrane Protein ◽  
Coiled Coil ◽  
Tryptophan Residue ◽  
Amino Acid Residues ◽  
Functional Roles ◽  
Amino Terminal ◽  
Membrane Spanning ◽  
In Cells

TMEM120A, a member of the Transmembrane protein 120 (TMEM120) family, has pivotal function in adipocyte differentiation and metabolism, and may also contribute to sensing mechanical pain by functioning as an ion channel named TACAN. Here we report that expression of TMEM120A is not sufficient in mediating poking- or stretch-induced currents in cells, and have solved cryo-EM structures of human TMEM120A (HsTMEM120A) in complex with an endogenous metabolic cofactor (coenzyme A, CoASH) and in the apo form. HsTMEM120A forms a symmetrical homodimer with each monomer containing an amino-terminal coiled-coil motif followed by a transmembrane domain with six membrane-spanning helices. Within the transmembrane domain, a CoASH molecule is hosted in a deep cavity and forms specific interactions with nearby amino acid residues. Mutation of a central tryptophan residue involved in binding CoASH dramatically reduced the binding affinity of HsTMEM120A with CoASH. HsTMEM120A exhibits distinct conformations at the states with or without CoASH bound. Our results suggest that TMEM120A may have alternative functional roles potentially involved in CoASH transport, sensing or metabolism.

scholarly journals TMEM120A contains a specific coenzyme A-binding site and might not mediate poking- or stretch-induced channel activities in cells

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Yao Rong ◽  
Jinghui Jiang ◽  
Yiwei Gao ◽  
Jianli Guo ◽  
Danfeng Song ◽  
...  
Keyword(s):  
Coenzyme A ◽  
Transmembrane Domain ◽  
Transmembrane Protein ◽  
Coiled Coil ◽  
Tryptophan Residue ◽  
Amino Acid Residues ◽  
Functional Roles ◽  
Amino Terminal ◽  
Membrane Spanning ◽  
In Cells

TMEM120A, a member of the Transmembrane protein 120 (TMEM120) family, has pivotal function in adipocyte differentiation and metabolism, and may also contribute to sensing mechanical pain by functioning as an ion channel named TACAN. Here we report that expression of TMEM120A is not sufficient in mediating poking- or stretch-induced currents in cells, and have solved cryo-EM structures of human TMEM120A (HsTMEM120A) in complex with an endogenous metabolic cofactor (coenzyme A, CoASH) and in the apo form. HsTMEM120A forms a symmetrical homodimer with each monomer containing an amino-terminal coiled-coil motif followed by a transmembrane domain with six membrane-spanning helices. Within the transmembrane domain, a CoASH molecule is hosted in a deep cavity and forms specific interactions with nearby amino acid residues. Mutation of a central tryptophan residue involved in binding CoASH dramatically reduced the binding affinity of HsTMEM120A with CoASH. HsTMEM120A exhibits distinct conformations at the states with or without CoASH bound. Our results suggest that TMEM120A may have alternative functional roles potentially involved in CoASH transport, sensing or metabolism.

scholarly journals The O-glycosylated Stalk Domain Is Required for Apical Sorting of Neurotrophin Receptors in Polarized MDCK Cells

1997 ◽  
Vol 139 (4) ◽  
pp. 929-940 ◽  
Author(s):  
Charles Yeaman ◽  
Annick H. Le Gall ◽  
Anne N. Baldwin ◽  
Laure Monlauzeur ◽  
Andre Le Bivic ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Structural Information ◽  
Mdck Cells ◽  
Soluble Form ◽  
Apical Plasma Membrane ◽  
Neurotrophin Receptors ◽  
Juxtamembrane Region ◽  
Stalk Domain ◽  
Membrane Spanning ◽  
Apical Sorting

Delivery of newly synthesized membrane-spanning proteins to the apical plasma membrane domain of polarized MDCK epithelial cells is dependent on yet unidentified sorting signals present in the luminal domains of these proteins. In this report we show that structural information for apical sorting of transmembrane neurotrophin receptors (p75NTR) is localized to a juxtamembrane region of the extracellular domain that is rich in O-glycosylated serine/threonine residues. An internal deletion of 50 amino acids that removes this stalk domain from p75NTR causes the protein to be sorted exclusively of the basolateral plasma membrane. Basolateral sorting stalk-minus p75NTR does not occur by default, but requires sequences present in the cytoplasmic domain. The stalk domain is also required for apical secretion of a soluble form of p75NTR, providing the first demonstration that the same domain can mediate apical sorting of both a membrane-anchored as well as secreted protein. However, the single N-glycan present on p75NTR is not required for apical sorting of either transmembrane or secreted forms.

scholarly journals Employing scan-SCAM to identify residues that compose transmembrane helix 1 (TM1) of EnvZ

2020 ◽  
Author(s):  
Thanwarat Chavalwan ◽  
Fadhael Alrahman H. Hasan ◽  
Phillipa Cheesman ◽  
Rahmi Yusuf ◽  
Roger R. Draheim
Keyword(s):  
Protein Interactions ◽  
Transmembrane Domain ◽  
Structural Information ◽  
Transmembrane Helix ◽  
Hydrophobic Core ◽  
Cytoplasmic Domains ◽  
Protein Protein Interactions ◽  
Diffusion Limited ◽  
Membrane Spanning ◽  
Intracellular Osmolarity

AbstractThe Escherichia coli sensor kinase EnvZ modulates porin expression in response to various stimuli including intracellular osmolarity, intracellular pH and periplasmic interaction with MzrA. The expression of two major outer membrane porins, OmpF and OmpC, are regulated by EnvZ, and act as passive diffusion-limited pores allowing compounds, including certain classes of antibiotics such as β-lactams and fluoroquinolones, to enter the bacterial cell. Even though allosteric processing occurs within both the periplasmic and cytoplasmic domains of EnvZ, how the transmembrane domain bi-directionally transmits these signals remains not fully understood. Here, we employ a library of single-Cys-containing EnvZ proteins to perform scan-SCAM in order to map the precise residue composition of TM1. Our results demonstrate that residue positions 19 through 30 reside within the membrane core and compose a tightly packed portion of TM1. We also show that positions 15 through 18 and position 31 are interfacial and slightly splayed apart compared to those tightly packed within the hydrophobic core. Finally, we reveal that residue positions 33 and 34 reside in the periplasm and participate in robust protein-protein interactions, while the periplasmic positions 35 through 41 exhibit helical periodicity. We conclude by synthesizing these new insights with recent high-resolution structural information into a model of membrane-spanning allosteric coupling between the periplasmic and cytoplasmic domains of EnvZ.

scholarly journals Localization of Sed5, a putative vesicle targeting molecule, to the cis-Golgi network involves both its transmembrane and cytoplasmic domains.

1994 ◽  
Vol 127 (2) ◽  
pp. 357-371 ◽  
Author(s):  
D K Banfield ◽  
M J Lewis ◽  
C Rabouille ◽  
G Warren ◽  
H R Pelham
Keyword(s):  
Plasma Membrane ◽  
Transmembrane Domain ◽  
Protein A ◽  
Similar Distribution ◽  
Cos Cells ◽  
Transport Vesicles ◽  
Membrane Spanning ◽  
Golgi Network ◽  
Vesicular Compartment

The yeast Sed5 protein, which is required for vesicular transport between ER and Golgi complex, is a membrane protein of the syntaxin family. These proteins are thought to provide the specific targets that are recognized by transport vesicles. We have investigated the mechanism by which Sed5 protein is itself localized. Expression of epitope-tagged versions of the yeast, Drosophila and rat Sed5 homologues in COS cells results in a perinuclear distribution; immuno-EM reveals that the majority of the protein is in a tubulo-vesicular compartment on the cis side of the Golgi apparatus. A similar distribution was obtained with a chimeric molecule consisting of a plasma membrane syntaxin with the Drosophila Sed5 transmembrane domain. This indicates that the membrane-spanning domain contains targeting information, as is the case with resident Golgi enzymes. However, alterations to the transmembrane domain of Drosophila Sed5 itself did not result in its mistargeting, implying that an additional targeting mechanism exists which involves only the cytoplasmic part of the protein. This was confirmed by modifying the transmembrane domain of the yeast Sed5 protein: substitution with the corresponding region from the Sso1 protein (a plasma membrane syntaxin homologue) did not affect yeast Sed5 function in vivo.

scholarly journals Contribution of the Cytoplasmic Determinants of Vpu to the Expansion of Virus-Containing Compartments in HIV-1-Infected Macrophages

2019 ◽  
Vol 93 (11) ◽  
Author(s):  
Olivier Leymarie ◽  
Leslie Lepont ◽  
Margaux Versapuech ◽  
Delphine Judith ◽  
Sophie Abelanet ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Viral Particle ◽  
Transmembrane Domain ◽  
Immune Surveillance ◽  
Restriction Factor ◽  
Viral Particles ◽  
Protein Functions ◽  
Specific Accumulation ◽  
Model Cell ◽  
Hiv 1

ABSTRACTHIV-1 infection of macrophages leads to the sequestration of newly formed viruses in intracellular plasma membrane-connected structures termed virus-containing compartments (VCCs), where virions remain infectious and hidden from immune surveillance. The cellular restriction factor bone marrow stromal cell antigen 2 (BST2), which prevents HIV-1 dissemination by tethering budding viral particles at the plasma membrane, can be found in VCCs. The HIV-1 accessory protein Vpu counteracts the restriction factor BST2 by downregulating its expression and removing it from viral budding sites. Numerous studies described these Vpu countermeasures in CD4+T cells or model cell lines, but the interplay between Vpu and BST2 in VCC formation and HIV-1 production in macrophages is less explored. Here, we show that Vpu expression in HIV-1-infected macrophages enhances viral release. This effect is related to Vpu’s ability to circumvent BST2 antiviral activity. We show that in absence of Vpu, BST2 is enriched in VCCs and colocalizes with capsid p24, whereas Vpu expression significantly reduces the presence of BST2 in these compartments. Furthermore, our data reveal that BST2 is dispensable for the formation of VCCs and that Vpu expression impacts the volume of these compartments. This Vpu activity partly depends on BST2 expression and requires the integrity of the Vpu transmembrane domain, the dileucine-like motif E59XXXLV64and phosphoserines 52 and 56 of Vpu. Altogether, these results highlight that Vpu controls the volume of VCCs and promotes HIV-1 release from infected macrophages.IMPORTANCEHIV-1 infection of macrophages leads to the sequestration of newly formed viruses in virus-containing compartments (VCCs), where virions remain infectious and hidden from immune surveillance. The restriction factor BST2, which prevents HIV-1 dissemination by tethering budding viral particles, can be found in VCCs. The HIV-1 Vpu protein counteracts BST2. This study explores the interplay between Vpu and BST2 in the viral protein functions on HIV-1 release and viral particle sequestration in VCCs in macrophages. The results show that Vpu controls the volume of VCCs and favors viral particle release. These Vpu functions partly depend on Vpu’s ability to antagonize BST2. This study highlights that the transmembrane domain of Vpu and two motifs of the Vpu cytoplasmic domain are required for these functions. These motifs were notably involved in the control of the volume of VCCs by Vpu but were dispensable for the prevention of the specific accumulation of BST2 in these structures.

scholarly journals TomoSAR Mapping of 3D Forest Structure: Contributions of L-Band Configurations

2021 ◽  
Vol 13 (12) ◽  
pp. 2255
Author(s):  
Matteo Pardini ◽  
Victor Cazcarra-Bes ◽  
Konstantinos Papathanassiou
Keyword(s):  
Information Content ◽  
Forest Structure ◽  
Structural Information ◽  
3D Structure ◽  
Physical Structure ◽  
Structure Mapping ◽  
Structure Information ◽  
Forest Sites ◽  
L Band ◽  
Structure Indices

Synthetic Aperture Radar (SAR) measurements are unique for mapping forest 3D structure and its changes in time. Tomographic SAR (TomoSAR) configurations exploit this potential by reconstructing the 3D radar reflectivity. The frequency of the SAR measurements is one of the main parameters determining the information content of the reconstructed reflectivity in terms of penetration and sensitivity to the individual vegetation elements. This paper attempts to review and characterize the structural information content of L-band TomoSAR reflectivity reconstructions, and their potential to forest structure mapping. First, the challenges in the accurate TomoSAR reflectivity reconstruction of volume scatterers (which are expected to dominate at L-band) and to extract physical structure information from the reconstructed reflectivity is addressed. Then, the L-band penetration capability is directly evaluated by means of the estimation performance of the sub-canopy ground topography. The information content of the reconstructed reflectivity is then evaluated in terms of complementary structure indices. Finally, the dependency of the TomoSAR reconstruction and of its structural information to both the TomoSAR acquisition geometry and the temporal change of the reflectivity that may occur in the time between the TomoSAR measurements in repeat-pass or bistatic configurations is evaluated. The analysis is supported by experimental results obtained by processing airborne acquisitions performed over temperate forest sites close to the city of Traunstein in the south of Germany.

Identification of a di-leucine motif within the C terminus domain of the Menkes disease protein that mediates endocytosis from the plasma membrane

1999 ◽  
Vol 112 (11) ◽  
pp. 1721-1732 ◽  
Author(s):  
M.J. Francis ◽  
E.E. Jones ◽  
E.R. Levy ◽  
R.L. Martin ◽  
S. Ponnambalam ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Golgi Apparatus ◽  
Transmembrane Domain ◽  
Menkes Disease ◽  
Cytoplasmic Domain ◽  
Endocytic Pathway ◽  
Site Directed Mutagenesis ◽  
Trans Golgi Network ◽  
C Terminus ◽  
Golgi Network

The protein encoded by the Menkes disease gene (MNK) is localised to the Golgi apparatus and cycles between the trans-Golgi network and the plasma membrane in cultured cells on addition and removal of copper to the growth medium. This suggests that MNK protein contains active signals that are involved in the retention of the protein to the trans-Golgi network and retrieval of the protein from the plasma membrane. Previous studies have identified a signal involved in Golgi retention within transmembrane domain 3 of MNK. To identify a motif sufficient for retrieval of MNK from the plasma membrane, we analysed the cytoplasmic domain, downstream of transmembrane domain 7 and 8. Chimeric constructs containing this cytoplasmic domain fused to the reporter molecule CD8 localised the retrieval signal(s) to 62 amino acids at the C terminus. Further studies were performed on putative internalisation motifs, using site-directed mutagenesis, protein expression, chemical treatment and immunofluorescence. We observed that a di-leucine motif (L1487L1488) was essential for rapid internalisation of chimeric CD8 proteins and the full-length Menkes cDNA from the plasma membrane. We suggest that this motif mediates the retrieval of MNK from the plasma membrane into the endocytic pathway, via the recycling endosomes, but is not sufficient on its own to return the protein to the Golgi apparatus. These studies provide a basis with which to identify other motifs important in the sorting and delivery of MNK from the plasma membrane to the Golgi apparatus.

Interaction of the endoplasmic reticulum α1,2-mannosidase Mns1p with Rer1p using the split-ubiquitin system

2001 ◽  
Vol 114 (24) ◽  
pp. 4629-4635
Author(s):  
Michel J. Massaad ◽  
Annette Herscovics
Keyword(s):  
Endoplasmic Reticulum ◽  
Catalytic Domain ◽  
Transmembrane Domain ◽  
Protein A ◽  
Yeast Cells ◽  
Type Ii ◽  
Ubiquitin System ◽  
Endoplasmic Reticulum Protein ◽  
Split Ubiquitin

The α1,2-mannosidase Mns1p involved in the N-glycosidic pathway in Saccharomyces cerevisiae is a type II membrane protein of the endoplasmic reticulum. The localization of Mns1p depends on retrieval from the Golgi through a mechanism that involves Rer1p. A chimera consisting of the transmembrane domain of Mns1p fused to the catalytic domain of the Golgi α1,2-mannosyltransferase Kre2p was localized in the endoplasmic reticulum of Δpep4 cells and in the vacuoles of rer1/Δpep4 by indirect immunofluorescence. The split-ubiquitin system was used to determine if there is an interaction between Mns1p and Rer1p in vivo. Co-expression of NubG-Mns1p and Rer1p-Cub-protein A-lexA-VP16 in L40 yeast cells resulted in cleavage of the reporter molecule, protein A-lexA-VP16, detected by western blot analysis and by expression of β-galactosidase activity. Sec12p, another endoplasmic reticulum protein that depends on Rer1p for its localization, also interacted with Rer1p using the split-ubiquitin assay, whereas the endoplasmic reticulum protein Ost1p showed no interaction. A weak interaction was observed between Alg5p and Rer1p. These results demonstrate that the transmembrane domain of Mns1p is sufficient for Rer1p-dependent endoplasmic reticulum localization and that Mns1p and Rer1p interact. Furthermore, the split-ubiquitin system demonstrates that the C-terminal of Rer1p is in the cytosol.

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