scholarly journals The Ftr1p iron permease in the yeast plasma membrane: orientation, topology and structure-function relationships

2004 ◽  
Vol 380 (2) ◽  
pp. 487-496 ◽  
Author(s):  
Scott SEVERANCE ◽  
Satadipta CHAKRABORTY ◽  
Daniel J. KOSMAN
Keyword(s):  
Plasma Membrane ◽  
Iron Uptake ◽  
Transmembrane Domain ◽  
Transmembrane Domains ◽  
Wild Type ◽  
Sequence Elements ◽  
Uptake Activity ◽  
Important Residue ◽  
Domain 3 ◽  
Double Substitution

Ftr1p is the permease component of the Fet3p–Ftr1p high affinity iron-uptake complex, in the plasma membrane of Saccharomyces cerevisiae, that transports the Fe3+ produced by the Fet3p ferroxidase into the cell. In this study we show that Ftr1p probably has seven transmembrane domains with an orientation of N-terminal outside, and C-terminal inside the cell. Within the context of this topology of the Fet3p–Ftr1p complex, we have identified several sequence elements in Ftr1p that are required for wild-type uptake function. First to be identified were two REXLE (Arg-Glu-Xaa-Leu-Glu) motifs in transmembrane domains 1 and 4. Alanine substitutions at any one of these combined six arginine or glutamic acid residues inactivated Ftr1p in iron uptake, indicating that both motifs were essential to iron permeation. R→K and E→D substitutions in these two motifs led to a variable loss of activity, suggesting that while all six residues were essential, their contributions to uptake were quantitatively and/or mechanistically distinct. The terminal glutamate in an EDLWE89 element, associated with transmembrane domain 3, and a DASE motif, located in extracellular loop 6, were also required. The double substitution to AASA in the latter, inactivated Ftr1p in iron uptake while the Ftr1p(E89A) mutant had only 20% of wild-type activity. The two REXLE and the EDLWE and DASE motifs are strongly conserved among fungal Ftr1p homologues, suggesting that these motifs are essential to iron permeation. Finally another important residue, Ile369, was identified in the Ftr1p cytoplasmic C-terminal domain. Deletion or substitution of this residue led to a 70% loss of iron-uptake activity. Ile369 was the only residue identified in this domain that made such a major contribution to iron uptake by the Fet3p–Ftr1p complex.

scholarly journals Regulation of Copper Metabolism by Nitrogen Utilization in Saccharomyces cerevisiae

2021 ◽  
Vol 7 (9) ◽  
pp. 756
Author(s):  
Suzie Kang ◽  
Hyewon Seo ◽  
Min-Gyu Lee ◽  
Cheol-Won Yun
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Iron Uptake ◽  
Nitrogen Starvation ◽  
Copper Metabolism ◽  
Nitrogen Utilization ◽  
Deletion Mutants ◽  
Uptake System ◽  
Wild Type ◽  
High Affinity ◽  
Uptake Activity

To understand the relationship between carbon or nitrogen utilization and iron homeostasis, we performed an iron uptake assay with several deletion mutants with partial defects in carbon or nitrogen metabolism. Among them, some deletion mutants defective in carbon metabolism partially and the MEP2 deletion mutant showed lower iron uptake activity than the wild type. Mep2 is known as a high-affinity ammonia transporter in Saccharomyces cerevisiae. Interestingly, we found that nitrogen starvation resulted in lower iron uptake activity than that of wild-type cells without downregulation of the genes involved in the high-affinity iron uptake system FET3/FTR1. However, the gene expression of FRE1 and CTR1 was downregulated by nitrogen starvation. The protein level of Ctr1 was also decreased by nitrogen starvation, and addition of copper to the nitrogen starvation medium partially restored iron uptake activity. However, the expression of MAC1, which is a copper-responsive transcriptional activator, was not downregulated by nitrogen starvation at the transcriptional level but was highly downregulated at the translational level. Mac1 was downregulated dramatically under nitrogen starvation, and treatment with MG132, which is an inhibitor of proteasome-dependent protein degradation, partially attenuated the downregulation of Mac1. Taken together, these results suggest that nitrogen starvation downregulates the high-affinity iron uptake system by degrading Mac1 in a proteasome-dependent manner and eventually downregulates copper metabolism.

Roles of the cytoplasmic and transmembrane domains of syntaxins in intracellular localization and trafficking

2001 ◽  
Vol 114 (17) ◽  
pp. 3115-3124 ◽  
Author(s):  
Kazuo Kasai ◽  
Kimio Akagawa
Keyword(s):  
Plasma Membrane ◽  
Transmembrane Domain ◽  
Intracellular Localization ◽  
Transmembrane Domains ◽  
Cytoplasmic Domains ◽  
Immunofluorescence Analysis ◽  
Attachment Protein ◽  
Transport Pathways ◽  
Sensitive Factor ◽  
Protein Receptors

Syntaxins are target-soluble N-ethylmaleimide-sensitive factor-attachment protein receptors (t-SNAREs) involved in docking and fusion of vesicles in exocytosis and endocytosis. Many syntaxin isoforms have been isolated, and each one displays a distinct intracellular localization pattern. However, the signals that drive the specific intracellular localization of syntaxins are poorly understood. In this study, we used indirect immunofluorescence analysis to examine the localization of syntaxin chimeras, each containing a syntaxin transmembrane domain fused to a cytoplasmic domain derived from a different syntaxin. We show that the cytoplasmic domains of syntaxins 5, 6, 7 and 8 have important effects on intracellular localization. We also demonstrate that the transmembrane domain of syntaxin 5 is sufficient to localize the chimera to the compartment expected for wild-type syntaxin 5. Additionally, we find that syntaxins 6, 7 and 8, but not syntaxin 5, are present at the plasma membrane, and that these syntaxins cycle through the plasma membrane by virtue of their cytoplasmic domains. Finally, we find that di-leucine-based motifs in the cytoplasmic domains of syntaxins 7 and 8 are necessary for their intracellular localization and trafficking via distinct transport pathways. Combined, these results suggest that both the cytoplasmic and the transmembrane domains play important roles in intracellular localization and trafficking of syntaxins.

Transmembrane domain length determines intracellular membrane compartment localization of syntaxins 3, 4, and 5

2001 ◽  
Vol 281 (1) ◽  
pp. C215-C223 ◽  
Author(s):  
Robert T. Watson ◽  
Jeffrey E. Pessin
Keyword(s):  
Amino Acids ◽  
Plasma Membrane ◽  
Amino Acid ◽  
Glucose Transporter ◽  
Transmembrane Domain ◽  
Transmembrane Domains ◽  
Snare Protein ◽  
Glut 4 ◽  
Golgi Localization ◽  
Syntaxin 3

Insulin recruits glucose transporter 4 (GLUT-4) vesicles from intracellular stores to the plasma membrane in muscle and adipose tissue by specific interactions between the vesicle membrane-soluble N-ethylmaleimide-sensitive factor attachment protein target receptor (SNARE) protein VAMP-2 and the target membrane SNARE protein syntaxin 4. Although GLUT-4 vesicle trafficking has been intensely studied, few have focused on the mechanism by which the SNAREs themselves localize to specific membrane compartments. We therefore set out to identify the molecular determinants for localizing several syntaxin isoforms, including syntaxins 3, 4, and 5, to their respective intracellular compartments (plasma membrane for syntaxins 3 and 4; cis-Golgi for syntaxin 5). Analysis of a series of deletion and chimeric syntaxin constructs revealed that the 17-amino acid transmembrane domain of syntaxin 5 was sufficient to direct the cis-Golgi localization of several heterologous reporter constructs. In contrast, the longer 25-amino acid transmembrane domain of syntaxin 3 was sufficient to localize reporter constructs to the plasma membrane. Furthermore, truncation of the syntaxin 3 transmembrane domain to 17 amino acids resulted in a complete conversion to cis-Golgi compartmentalization that was indistinguishable from syntaxin 5. These data support a model wherein short transmembrane domains (≤17 amino acids) direct the cis-Golgi localization of syntaxins, whereas long transmembrane domains (≥23 amino acids) direct plasma membrane localization.

scholarly journals Elimination of the O-linked glycosylation site at Thr 104 results in the generation of a soluble human-transferrin receptor

Blood ◽  
1994 ◽  
Vol 83 (2) ◽  
pp. 580-586 ◽  
Author(s):  
EA Rutledge ◽  
BJ Root ◽  
JJ Lucas ◽  
CA Enns
Keyword(s):  
Plasma Membrane ◽  
Amino Acid ◽  
Transferrin Receptor ◽  
Transmembrane Domain ◽  
Glycosylation Site ◽  
Soluble Form ◽  
Protein Sequence Analysis ◽  
Wild Type ◽  
Human Transferrin Receptor ◽  
Intracellular Compartments

The transferrin receptor (TfR) is the plasma membrane protein responsible for the binding and internalization of the major iron- transport protein, transferrin. The function of the single O-linked oligosaccharide near the transmembrane domain of the TfR at amino acid Thr 104 is unknown. To elucidate the effect of the O-linked carbohydrate on TfR function, the oligosaccharide was eliminated by replacing Thr 104 with Asp and the mutated cDNA was expressed in a cell line lacking endogenous TfR. Elimination of the oligosaccharide at Thr 104 results in a form of the receptor that is susceptible to cleavage. A 78-kD soluble TfR that can bind transferrin is released into the growth medium. The intact mutant TfR is not grossly altered in its structure and does not differ significantly from the wild-type human receptor in many respects: (1) It shows the same distribution between the plasma membrane and intracellular compartments; (2) the binding constant for transferrin is similar to that of the wild-type TfR; and (3) it is not rapidly degraded. Protein-sequence analysis of the soluble form indicates that the sequence begins at amino acid 101 of the intact receptor. This is the same cleavage site reported for a soluble form of normal receptor found in human serum. Substitution of Gly, Glu, or Met at position 104 also results in increased cleavage of the TfR and suggests that elimination of the O-linked carbohydrate at position 104 enhances the susceptibility of TfR to cleavage and may mimic a naturally occurring process previously described as being related to erythropoiesis.

scholarly journals Mutational Analysis of the Human Immunodeficiency Virus Type 1 Vpu Transmembrane Domain That Promotes the Enhanced Release of Virus-Like Particles from the Plasma Membrane of Mammalian Cells

1998 ◽  
Vol 72 (2) ◽  
pp. 1270-1279 ◽  
Author(s):  
Mousumi Paul ◽  
Suparna Mazumder ◽  
Nicholas Raja ◽  
M. Abdul Jabbar
Keyword(s):  
Amino Acids ◽  
Human Immunodeficiency Virus ◽  
Plasma Membrane ◽  
Human Immunodeficiency Virus Type ◽  
Hela Cells ◽  
Mammalian Cells ◽  
Transmembrane Domain ◽  
Wild Type ◽  
Immunodeficiency Virus

ABSTRACT Human immunodeficiency virus type 1 Vpu is a multifunctional phosphoprotein composed of the N-terminal transmembrane (VpuTM) and C-terminal cytoplasmic domains. Each of these domains regulates a distinct function of the protein; the transmembrane domain is critical in virus release, and phosphorylation of the cytoplasmic domain is necessary for CD4 proteolysis. We carried our experiments to identify amino acids in the VpuTM domain that are important in the process of virus-like particle (VLP) release from HeLa cells. VLPs are released from the plasma membrane of HeLa cells at constitutive levels, and Vpu expression enhanced the release of VLPs by a factor of 10 to 15. Deletion of two to five amino acids from both N- and C-terminal ends or the middle of the VpuTM domain generated mutant Vpu proteins that have lost the ability to enhance VLP release. These deletion mutants have not lost the ability to associate with the wild-type or mutant Vpu proteins and formed complexes with equal efficiency. They were also transported normally to the Golgi complex. Furthermore, a Vpu protein having the CD4 transmembrane and Vpu cytoplasmic domains was completely inactive, and Vpu proteins harboring hybrid Vpu-CD4 TM domains were also defective in the ability to enhance the release of VLPs. When tested for functional complementation in cotransfected cells, two inactive proteins were not able to reconstitute Vpu activity that enhances the release of Gag particles. Coexpression of functional CD4/Vpu hybrids or wild-type Vpu with inactive mutant CD4/Vpu proteins revealed that mutations in the VpuTM domain could dominantly interfere with Vpu activity in Gag release. Taken together, these results demonstrated that the structural integrity of the VpuTM domain is critical for Vpu activity in the release of VLPs from the plasma membrane of mammalian cells.

scholarly journals Important Role for the Transmembrane Domain of Severe Acute Respiratory Syndrome Coronavirus Spike Protein during Entry

2006 ◽  
Vol 80 (3) ◽  
pp. 1302-1310 ◽  
Author(s):  
Rene Broer ◽  
Bertrand Boson ◽  
Willy Spaan ◽  
François-Loïc Cosset ◽  
Jeroen Corver
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Membrane Fusion ◽  
Cell Fusion ◽  
Transmembrane Domain ◽  
Cytoplasmic Tail ◽  
Transmembrane Domains ◽  
Spike Protein ◽  
Fusion Activity ◽  
Wild Type ◽  
Cell Cell

ABSTRACT The spike protein (S) of severe acute respiratory syndrome coronavirus (SARS-CoV) is responsible for receptor binding and membrane fusion. It contains a highly conserved transmembrane domain that consists of three parts: an N-terminal tryptophan-rich domain, a central domain, and a cysteine-rich C-terminal domain. The cytoplasmic tail of S has previously been shown to be required for assembly. Here, the roles of the transmembrane and cytoplasmic domains of S in the infectivity and membrane fusion activity of SARS-CoV have been studied. SARS-CoV S-pseudotyped retrovirus (SARSpp) was used to measure S-mediated infectivity. In addition, the cell-cell fusion activity of S was monitored by a Renilla luciferase-based cell-cell fusion assay. Svsv-cyt, an S chimera with a cytoplasmic tail derived from vesicular stomatitis virus G protein (VSV-G), and Smhv-tmdcyt, an S chimera with the cytoplasmic and transmembrane domains of mouse hepatitis virus, displayed wild-type-like activity in both assays. Svsv-tmdcyt, a chimera with the cytoplasmic and transmembrane domains of VSV-G, was impaired in the SARSpp and cell-cell fusion assays, showing 3 to 25% activity compared to the wild type, depending on the assay and the cells used. Examination of the oligomeric state of the chimeric S proteins in SARSpp revealed that Svsv-tmdcyt trimers were less stable than wild-type S trimers, possibly explaining the lowered fusogenicity and infectivity.

scholarly journals Functional and mechanistic roles of the human proton-coupled folate transporter transmembrane domain 6–7 linker

2016 ◽  
Vol 473 (20) ◽  
pp. 3545-3562 ◽  
Author(s):  
Mike R. Wilson ◽  
Zhanjun Hou ◽  
Lucas J. Wilson ◽  
Jun Ye ◽  
Larry H. Matherly
Keyword(s):  
Secondary Structure ◽  
Targeted Delivery ◽  
Transmembrane Domain ◽  
Surface Expression ◽  
Chimeric Proteins ◽  
Transport Activity ◽  
Wild Type ◽  
Intracellular Loop ◽  
Mutant Proteins ◽  
Sequence Elements

The proton-coupled folate transporter (PCFT; SLC46A1) is a folate–proton symporter expressed in solid tumors and is used for tumor-targeted delivery of cytotoxic antifolates. Topology modeling suggests that the PCFT secondary structure includes 12 transmembrane domains (TMDs) with TMDs 6 and 7 linked by an intracellular loop (positions 236–265) including His247, implicated as functionally important. Single-cysteine (Cys) mutants were inserted from positions 241 to 251 in Cys-less PCFT and mutant proteins were expressed in PCFT-null (R1-11) HeLa cells; none were reactive with 2-aminoethyl methanethiosulfonate biotin, suggesting that the TMD6–7 loop is intracellular. Twenty-nine single alanine mutants spanning the entire TMD6–7 loop were expressed in R1-11 cells; activity was generally preserved, with the exception of the 247, 250, and 251 mutants, partly due to decreased surface expression. Coexpression of PCFT TMD1–6 and TMD7–12 half-molecules in R1-11 cells partially restored transport activity, although removal of residues 252–265 from TMD7–12 abolished transport. Chimeric proteins, including a nonhomologous sequence from a thiamine transporter (ThTr1) inserted into the PCFT TMD6–7 loop (positions 236–250 or 251–265), were active, although replacement of the entire loop with the ThTr1 sequence resulted in substantial loss of activity. Amino acid replacements (Ala, Arg, His, Gln, and Glu) or deletions at position 247 in wild-type and PCFT–ThTr1 chimeras resulted in differential effects on transport. Collectively, our findings suggest that the PCFT TMD6–7 connecting loop confers protein stability and may serve a unique functional role that depends on secondary structure rather than particular sequence elements.

scholarly journals The Cytoplasmic End of Transmembrane Domain 3 Regulates the Activity of the Saccharomyces cerevisiae G-Protein-Coupled α-Factor Receptor

Genetics ◽  
2002 ◽  
Vol 160 (2) ◽  
pp. 429-443
Author(s):  
William Parrish ◽  
Markus Eilers ◽  
Weiwen Ying ◽  
James B Konopka
Keyword(s):  
G Protein ◽  
Transmembrane Domain ◽  
Transmembrane Helix ◽  
G Protein Coupled Receptors ◽  
Transmembrane Domains ◽  
Targeted Mutagenesis ◽  
Activating Mutations ◽  
Polar Residues ◽  
Domain 3 ◽  
G Protein Coupled

Abstract The binding of α-factor to its receptor (Ste2p) activates a G-protein-signaling pathway leading to conjugation of MATa cells of the budding yeast S. cerevisiae. We conducted a genetic screen to identify constitutively activating mutations in the N-terminal region of the α-factor receptor that includes transmembrane domains 1–5. This approach identified 12 unique constitutively activating mutations, the strongest of which affected polar residues at the cytoplasmic ends of transmembrane domains 2 and 3 (Asn84 and Gln149, respectively) that are conserved in the α-factor receptors of divergent yeast species. Targeted mutagenesis, in combination with molecular modeling studies, suggested that Gln149 is oriented toward the core of the transmembrane helix bundle where it may be involved in mediating an interaction with Asn84. These residues appear to play specific roles in maintaining the inactive conformation of the protein since a variety of mutations at either position cause constitutive receptor signaling. Interestingly, the activity of many mammalian G-protein-coupled receptors is also regulated by conserved polar residues (the E/DRY motif) at the cytoplasmic end of transmembrane domain 3. Altogether, the results of this study suggest a conserved role for the cytoplasmic end of transmembrane domain 3 in regulating the activity of divergent G-protein-coupled receptors.

Conserved residues F316 and G476 in the concentrative nucleoside transporter 1 (hCNT1) affect guanosine sensitivity and membrane expression, respectively

2005 ◽  
Vol 288 (1) ◽  
pp. C39-C45 ◽  
Author(s):  
Yurong Lai ◽  
Eun-Woo Lee ◽  
Carl C. Ton ◽  
Shashi Vijay ◽  
Huixia Zhang ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Fluorescent Protein ◽  
Transmembrane Domain ◽  
Mdck Cells ◽  
Site Directed Mutagenesis ◽  
Nucleoside Transporter ◽  
Wild Type ◽  
Membrane Expression ◽  
Concentrative Nucleoside Transporter ◽  
Green Fluorescent

The functional significance of two highly conserved amino acid residues, F316 [putative transmembrane domain (TM)7] and G476 (putative TM11), in the concentrative nucleoside transporter hCNT1 (SLC28A1) was examined by performing site-directed mutagenesis. Conservative mutations at these positions (F316A, F316Y, G476A, and G476L) were generated and expressed in Madin-Darby canine kidney (MDCK) cells as fusion polypeptides with green fluorescent protein (GFP). Unlike wild-type hCNT1, G476A-GFP and G476L-GFP were not expressed in the plasma membrane in undifferentiated or differentiated MDCK cells and had no functional activity. Like wild-type hCNT1, F316A-GFP and F316Y-GFP were expressed in the plasma membrane of undifferentiated MDCK cells and in the apical membrane of differentiated MDCK cells. Remarkably, transport of [3H]uridine by F316Y-GFP or F316A-GFP was highly sensitive to inhibition by guanosine. Furthermore, genotyping of exon 11 of hCNT1 (TM7) in a panel of 260 anonymous human DNA samples revealed a novel F316H variant (TT>CA; 1/260). When expressed in MDCK cells, [3H]uridine transport by F316H was also found to be sensitive to inhibition by guanosine (IC50 = 148 μM). The effect of the F316H mutation resembles the N4 type nucleoside transporter phenotype previously reported to be present in human kidneys. We suggest that the N4 transport system is a naturally occurring variant of hCNT1, perhaps at the F316 position. Collectively, our data show that G476 is important for correct membrane targeting, folding, and/or intracellular processing of hCNT1. In addition, we have discovered that hCNT1 displays natural variation at position F316 and that the variant F316H confers on the transporter an unusual sensitivity to inhibition by guanosine.

scholarly journals Genetic evidence that ferric reductase is required for iron uptake in Saccharomyces cerevisiae.

1990 ◽  
Vol 10 (5) ◽  
pp. 2294-2301 ◽  
Author(s):  
A Dancis ◽  
R D Klausner ◽  
A G Hinnebusch ◽  
J G Barriocanal
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Plasma Membrane ◽  
Iron Uptake ◽  
Reductase Activity ◽  
Gene Transcript ◽  
Single Mutation ◽  
Uptake System ◽  
Ferric Reductase ◽  
Wild Type ◽  
Iron Starvation

The requirement for a reduction step in cellular iron uptake has been postulated, and the existence of plasma membrane ferric reductase activity has been described in both procaryotic and eucaryotic cells. In the yeast Saccharomyces cerevisiae, there is an externally directed reductase activity that is regulated by the concentration of iron in the growth medium; maximal activity is induced by iron starvation. We report here the isolation of a mutant of S. cerevisiae lacking the reductase activity. This mutant is deficient in the uptake of ferric iron and is extremely sensitive to iron deprivation. Genetic analysis of the mutant demonstrates that the reductase and ferric uptake deficiencies are due to a single mutation that we designate fre1-1. Both phenotypes cosegregate in meiosis, corevert with a frequency of 10(-7), and are complemented by a 3.5-kilobase fragment of genomic DNA from wild-type S. cerevisiae. This fragment contains FRE1, the wild-type allele of the mutant gene. The level of the gene transcript is regulated by iron in the same was as the reductase activity. The ferrous ion product of the reductase must traverse the plasma membrane. A high-affinity (Km = 5 microM) ferrous uptake system is present in both wild-type and mutant cells. Thus, iron uptake in S. cerevisiae is mediated by two plasma membrane components, a reductase and a ferrous transport system.

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