Characterization of nuclear pore complex targeting domains in Pom152 in Saccharomyces cerevisiae

Biology Open ◽

10.1242/bio.057661 ◽

2021 ◽

Author(s):

Jacqueline T. Brown ◽

Alexandra J. Haraczy ◽

Christopher M. Wilhelm ◽

Kenneth D. Belanger

Keyword(s):

Amino Acids ◽

Nuclear Pore Complex ◽

Transmembrane Domain ◽

Transmembrane Protein ◽

Full Length ◽

Nuclear Pore ◽

Amino Terminal ◽

Glycosylation Sites ◽

Cytosolic Side ◽

Carboxy Terminal

Pom152 is a transmembrane protein within the nuclear pore complex (NPC) of fungi that is important for NPC assembly and structure. Pom152 is comprised of a short amino-terminal region that remains on the cytosolic side of the nuclear envelope (NE) and interacts with NPC proteins, a transmembrane domain, and a large, glycosylated carboxy-terminal domain within the NE lumen. Here we show that the N-terminal 200 amino acids of Pom152 that include only the amino-terminal and transmembrane regions are sufficient for localization to the NPC. Full-length, glycosylation-deficient, and truncated Pom152-GFP chimeras expressed in cells containing endogenous Pom152 localize to both NPCs and cortical endoplasmic reticulum (ER). Expression of Pom152-GFP fusions in pom152Δ cells results in detectable localization at only the NE by full-length and amino-terminal Pom152-GFP fusions, but continued retention at both the NE and ER for a chimera lacking just the carboxy-terminal 377 amino acids. Neither deletion of Pom152 nor its carboxy-terminal glycosylation sites altered the nuclear protein export rate of an Msn5/Kap142 protein cargo. These data narrow the Pom152 region sufficient for NPC localization and provide evidence that alterations in other domains may impact Pom152 targeting or affinity for the NPC.

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nup1 mutants exhibit pleiotropic defects in nuclear pore complex function.

The Journal of Cell Biology ◽

10.1083/jcb.127.2.319 ◽

1994 ◽

Vol 127 (2) ◽

pp. 319-332 ◽

Cited By ~ 45

Author(s):

A M Bogerd ◽

J A Hoffman ◽

D C Amberg ◽

G R Fink ◽

L I Davis

Keyword(s):

Nuclear Envelope ◽

Nuclear Pore Complex ◽

Structural Integrity ◽

Mutational Analysis ◽

Complex Function ◽

Spindle Pole ◽

Nuclear Pore ◽

Temperature Sensitive ◽

Amino Terminal ◽

Carboxy Terminal

The NUP1 gene of Saccharomyces cerevisiae encodes one member of a family of nuclear pore complex proteins (nucleoporins) conserved from yeast to vertebrates. We have used mutational analysis to investigate the function of Nup1p. Deletion of either the amino- or carboxy-terminal domain confers a lethal phenotype, but partial truncations at either end affect growth to varying extents. Amino-terminal truncation causes mislocalization and degradation of the mutant protein, suggesting that this domain is required for targeting Nup1p to the nuclear pore complex. Carboxy-terminal mutants are stable but do not have wild-type function, and confer a temperature sensitive phenotype. Both import of nuclear proteins and export of poly(A) RNA are defective at the nonpermissive temperature. In addition, nup1 mutant cells become multinucleate at all temperatures, a phenotype suggestive of a defect in nuclear migration. Tubulin staining revealed that the mitotic spindle appears to be oriented randomly with respect to the bud, in spite of the presence of apparently normal cytoplasmic microtubules connecting one spindle pole body to the bud tip. EM analysis showed that the nuclear envelope forms long projections extending into the cytoplasm, which appear to have detached from the bulk of the nucleus. Our results suggest that Nup1p may be required to retain the structural integrity between the nuclear envelope and an underlying nuclear scaffold, and that this connection is required to allow reorientation of the nucleus in response to cytoskeletal forces.

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Autoantibodies from patients with primary biliary cirrhosis recognize a restricted region within the cytoplasmic tail of nuclear pore membrane glycoprotein Gp210.

Journal of Experimental Medicine ◽

10.1084/jem.178.6.2237 ◽

1993 ◽

Vol 178 (6) ◽

pp. 2237-2242 ◽

Cited By ~ 64

Author(s):

R E Nickowitz ◽

H J Worman

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Primary Biliary Cirrhosis ◽

Antigenic Determinants ◽

Nuclear Pore ◽

Biliary Cirrhosis ◽

Amino Terminal ◽

Terminal Domain ◽

Carboxy Terminal ◽

Amino Terminal Domain

Patients with primary biliary cirrhosis (PBC) frequently have autoantibodies against a 210-kD integral glycoprotein of the nuclear envelope pore membrane. This protein, termed gp210, has a 1,783-amino acid amino-terminal domain located in the perinuclear space, a 20-amino acid transmembrane segment, and a 58-amino acid cytoplasmic carboxy-terminal tail. We now demonstrate that autoantibodies from 25 patients with PBC that recognize gp210 react with the cytoplasmic carboxy-terminal tail while none react with unmodified linear epitopes in the amino-terminal domain. The epitope(s) recognized by autoantibodies from all 25 patients is contained within a stretch of 15 amino acids. The recognized amino acid sequence is homologous to the protein products of the Escherichia coli mutY gene and Salmonella typhimurium mutB gene with an exact identity of six consecutive amino acids, suggesting that anti-gp210 antibodies may arise by molecular mimicry of bacterial antigenic determinants.

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

10.1101/2021.06.17.448797 ◽

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.

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

eLife ◽

10.7554/elife.71474 ◽

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.

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Tpr, a large coiled coil protein whose amino terminus is involved in activation of oncogenic kinases, is localized to the cytoplasmic surface of the nuclear pore complex.

The Journal of Cell Biology ◽

10.1083/jcb.127.6.1515 ◽

1994 ◽

Vol 127 (6) ◽

pp. 1515-1526 ◽

Cited By ~ 77

Author(s):

D A Byrd ◽

D J Sweet ◽

N Panté ◽

K N Konstantinov ◽

T Guan ◽

...

Keyword(s):

Amino Acids ◽

Rat Liver ◽

Nuclear Pore Complex ◽

Protein Import ◽

Coiled Coil ◽

In Vitro Translation ◽

Immunogold Electron Microscopy ◽

Nuclear Pore ◽

Amino Terminal ◽

Cytoplasmic Surface

From a panel of monoclonal antibodies raised against fractions of rat liver nuclear envelopes (NEs), we have identified an antibody, RL30, which reacts with novel nuclear pore complex (NPC) antigens that are not O-glycosylated. By immunofluorescence staining of cultured cells, RL30 reacts exclusively with the NE in a punctate pattern that largely coincides with that of identified NPC proteins. RL30 labels only the cytoplasmic surface of the NPC in immunogold electron microscopy, predominantly in peripheral regions nearby the cytoplasmic ring. In immunoblots of isolated rat liver NEs and cultured rat cells, RL30 recognizes a 265-kD band, as well as a series of 175-265-kD bands in rat liver NEs that are likely to be proteolytic products of p265. Sequencing of peptides from the 175- and 265-kD RL30 antigens of rat liver revealed that they are both closely related to human Tpr, a protein whose amino-terminal 150-250 amino acids appear in oncogenic fusions with the kinase domains of the met, trk, and raf protooncogenes. We found that in vitro translation of human Tpr mRNA yields a major 265-kD band. Considered together, these data indicate that the 265-kD RL30 antigen in the NPC is the rat homologue of Tpr. Interestingly, Tpr contains an exceptionally long predicted coiled coil domain (approximately 1600 amino acids). The localization and predicted structure of Tpr suggest that it is a component of the cytoplasmic fibrils of the NPC implicated in nuclear protein import. Immunofluorescence microscopy shows that during NPC reassembly at the end of mitosis, Tpr becomes concentrated at the NE significantly later than O-linked glycoproteins, including p62. This indicates that reassembly of the NPC after mitosis is a stepwise process, and that the Tpr-containing peripheral structures are assembled later than p62.

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Multiple independent inputs are required for activation of the p70 S6 kinase.

Molecular and Cellular Biology ◽

10.1128/mcb.15.5.2333 ◽

1995 ◽

Vol 15 (5) ◽

pp. 2333-2340 ◽

Cited By ~ 172

Author(s):

Q P Weng ◽

K Andrabi ◽

M T Kozlowski ◽

J R Grove ◽

J Avruch

Keyword(s):

Amino Acids ◽

Kinase Activity ◽

Phosphorylation Site ◽

Full Length ◽

S6 Kinase ◽

P70 S6 Kinase ◽

Amino Terminal ◽

Multisite Phosphorylation ◽

Carboxy Terminal

Previous studies have shown that the noncatalytic carboxy-terminal tail of the p70 S6 kinase (amino acids 422 to 525) contains an autoinhibitory pseudosubstrate domain that is phosphorylated in situ during activation and in vitro by mitogen-activated protein kinases. The present study shows that a recombinant p70 deleted of the carboxy-terminal tail (p70 delta CT104) nevertheless exhibits a basal and serum-stimulated 40S kinase activity and susceptibility to inhibition by wortmannin very similar to those of the parent, full-length p70 kinase. Carboxy-terminal deletion reduces the extent of maximal inhibition produced by rapamycin, from > 95% in the full-length p70 to 60 to 80% in p70 delta CT104, without altering the sensitivity to rapamycin inhibition (50% inhibitory concentration of 2 nM). Serum activation of p70 delta CT104, as with the parent, full-length p70, is accompanied by an increase in 32P content (about twofold) in situ and a slowing in electrophoretic mobility; both modifications are inhibited by pretreatment with wortmannin or rapamycin. 32P-peptide maps of p70 delta CT104 show multisite phosphorylation, and wortmannin and rapamycin appear to cause preferential dephosphorylation of the same subset of sites. Thus, it is likely that activation of the kinase requires phosphorylation of p70 at sites in addition to those previously identified in the carboxy-terminal tail. Evidence that the carboxy-terminal tail actually functions as a potent intramolecular inhibitor of kinase activity in situ is uncovered by deletion of a short acidic segment (amino acids 29 to 46) from the p70 amino-terminal noncatalytic region. Deletion of amino acids 29 to 46 causes a >95% inhibition of p70 activity despite continue phosphorylation of the carboxy-terminal tail in situ; additional deletion of the carboxy-terminal tail (yielding p70 delta 29-46/ delta CT104) increases activity 10-fold, to a level approaching that of p70 delta CT104. Deletion of residues 29 to 46 also abolishes completely the sensitivity of p70 to inhibition by rapamycin but does not alter the susceptibility to activation by serum of inhibition by wortmannin. Although the mechanisms underlying the effects of the delta 29-46 deletion are not known, they are not attributable to loss of the major in situ p70 phosphorylation site at Ser-40. Thus, activation of the p70 S6 kinase involves multiple, independent inputs directed at different domains of the p70 polypeptide. Disinhibition from the carboxy-terminal tail requires, in addition to its multisite phosphorylation, an activating input dependent on the presence of amino acids 29 to 46; this p70-activating input may be the same as that inhibited by rapamycin but is distinct from that arising from the wortmannin-inhibitable phosphatidylinositol 3-kinase. In addition, as exemplified by the rapamycin-resistant but mitogen- and wortmannin-sensitive p70 delta 29-46/ delta CT104 mutant, a further activating input, which probably involves site-specific phosphorylation in the segment between amino acids 46 to 421, is necessary.

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Expression of a Truncated Yeast Ccc1 Vacuolar Transporter Increases the Accumulation of Endogenous Iron

Genes ◽

10.3390/genes12081120 ◽

2021 ◽

Vol 12 (8) ◽

pp. 1120

Author(s):

Raquel Sorribes-Dauden ◽

María Teresa Martínez-Pastor ◽

Sergi Puig

Keyword(s):

Iron Homeostasis ◽

Sufficient Conditions ◽

Constitutive Expression ◽

Iron Bioavailability ◽

Full Length ◽

Yeast Cells ◽

Deficiency Anemia ◽

Functional Protein ◽

Amino Terminal ◽

In Cells

Iron is an essential micronutrient for all eukaryotic organisms because it participates as a redox cofactor in multiple metabolic processes. Iron bioavailability is highly restricted due to the low solubility of its oxidized form, frequently leading to iron deficiency anemia. The baker’s yeast Saccharomyces cerevisiae is used as a model organism for iron homeostasis studies, but also as a food supplement and fermentative microorganism in the food industry. Yeast cells use the vacuolar Ccc1 transporter to detoxify and store excess iron in the vacuoles. Here, we modulate CCC1 expression and properties to increase iron extraction from the environment. We show that constitutive expression of full-length CCC1 is toxic, whereas deletion of its cytosolic amino-terminal (Nt) domain (NtDCCC1) rescues this phenotype. Toxicity is exacerbated in cells lacking AFT1 transcription factor. Further characterization of NtDCcc1 protein suggests that it is a partially functional protein. Western blot analyses indicate that deletion of Ccc1 Nt domain does not significantly alter GFP-Ccc1 protein stability. A functional full-length GFP-Ccc1 protein localized to particular regions of the vacuolar membrane, whereas GFP-NtDCcc1 protein was evenly distributed throughout this endogenous membrane. Interestingly, expression of NtDCCC1 increased the accumulation of endogenous iron in cells cultivated under iron-sufficient conditions, a strategy that could be used to extract iron from media that are not rich in iron.

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Disruption of dynamic cell surface architecture of NIH3T3 fibroblasts by the N-terminal domains of moesin and ezrin: in vivo imaging with GFP fusion proteins

Journal of Cell Science ◽

10.1242/jcs.112.1.111 ◽

1999 ◽

Vol 112 (1) ◽

pp. 111-125 ◽

Cited By ~ 7

Author(s):

M.R. Amieva ◽

P. Litman ◽

L. Huang ◽

E. Ichimaru ◽

H. Furthmayr

Keyword(s):

Plasma Membrane ◽

Cell Surface ◽

Fluorescent Protein ◽

Cultured Cells ◽

Full Length ◽

Cell Behavior ◽

Amino Terminal ◽

Terminal Domain ◽

Carboxy Terminal Domain ◽

Carboxy Terminal

Lamellipodia, filopodia, microspikes and retraction fibers are characteristic features of a dynamic and continuously changing cell surface architecture and moesin, ezrin and radixin are thought to function in these microextensions as reversible links between plasma membrane proteins and actin microfilaments. Full-length and truncated domains of the three proteins were fused to green fluorescent protein (GFP), expressed in NIH3T3 cells, and distribution and behaviour of cells were analysed by using digitally enhanced differential interference contrast (DIC) and fluorescence video microscopy. The amino-terminal (N-)domains of all three proteins localize to the plasma membrane and fluorescence recordings parallel the dynamic changes in cell surface morphology observed by DIC microscopy of cultured cells. Expression of this domain, however, significantly affects cell surface architecture by the formation of abnormally long and fragile filopodia that poorly attach and retract abnormally. Even more striking are abundant irregular, branched and motionless membraneous structures that accumulate during retraction of lamellipodia. These are devoid of actin, endogenous moesin, ezrin and radixin, but contain the GFP-labeled domain. While a large proportion of endogenous proteins can be extracted with non-ionic detergents as in untransfected control cells, >90% of N-moesin and >60% of N-ezrin and N-radixin remain insoluble. The minimal size of the domain of moesin required for membrane localization and change in behavior includes residues 1–320. Deletions of amino acid residues from either end result in diffuse intracellular distribution, but also in normal cell behavior. Expression of GFP-fusions of full-length moesin or its carboxy-terminal domain has no effect on cell behavior during the observation period of 6–8 hours. The data suggest that, in the absence of the carboxy-terminal domain, N-moesin, -ezrin and -radixin interact tightly with the plasma membrane and interfere with normal functions of endogeneous proteins mainly during retraction.

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Functional Analysis of Tpr: Identification of Nuclear Pore Complex Association and Nuclear Localization Domains and a Role in mRNA Export

The Journal of Cell Biology ◽

10.1083/jcb.143.7.1801 ◽

1998 ◽

Vol 143 (7) ◽

pp. 1801-1812 ◽

Cited By ~ 73

Author(s):

Peter Bangs ◽

Brian Burke ◽

Christine Powers ◽

Roger Craig ◽

Aruna Purohit ◽

...

Keyword(s):

Nuclear Localization ◽

Nuclear Pore Complex ◽

Ectopic Expression ◽

Coiled Coil ◽

Full Length ◽

Nuclear Localization Sequence ◽

Nuclear Pore ◽

Nuclear Interior ◽

Mammalian Cell Lines ◽

Localization Sequence

Tpr is a 270-kD coiled-coil protein localized to intranuclear filaments of the nuclear pore complex (NPC). The mechanism by which Tpr contributes to the structure and function of the nuclear pore is currently unknown. To gain insight into Tpr function, we expressed the full-length protein and several subdomains in mammalian cell lines and examined their effects on nuclear pore function. Through this analysis, we identified an NH2-terminal domain that was sufficient for association with the nucleoplasmic aspect of the NPC. In addition, we unexpectedly found that the acidic COOH terminus was efficiently transported into the nuclear interior, an event that was apparently mediated by a putative nuclear localization sequence. Ectopic expression of the full-length Tpr caused a dramatic accumulation of poly(A)+ RNA within the nucleus. Similar results were observed with domains that localized to the NPC and the nuclear interior. In contrast, expression of these proteins did not appear to affect nuclear import. These data are consistent with a model in which Tpr is tethered to intranuclear filaments of the NPC by its coiled coil domain leaving the acidic COOH terminus free to interact with soluble transport factors and mediate export of macromolecules from the nucleus.

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