Proline residues in two tightly coupled helices of the sulphate transporter, SHST1, are important for sulphate transport

2001 ◽  
Vol 356 (2) ◽  
pp. 589-594 ◽  
Author(s):  
Megan C. SHELDEN ◽  
Patrick LOUGHLIN ◽  
M. Louise TIERNEY ◽  
Susan M. HOWITT
Keyword(s):  
Plasma Membrane ◽  
Critical Role ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Transmembrane Helices ◽  
Transport Activity ◽  
Post Translational Modification ◽  
Sulphate Transport ◽  
Stylosanthes Hamata ◽  
Tightly Coupled

The sulphate transporter SHST1, from Stylosanthes hamata, features three tightly coupled transmembrane helices which include proline residues that are conserved in most related transporters. We used site-directed mutagenesis and expression of the mutant transporters in yeast to test whether these proline residues are important for function. Four proline residues were replaced by both alanine and leucine. Only one of these proline residues, Pro-144, was essential for sulphate transport. However, mutation of either Pro-133 or Pro-160 resulted in a severe decrease in sulphate transport activity; this was due more to a decrease in transport activity than to a decrease in the amount of mutant SHST1 in the plasma membrane. These results suggest that all three proline residues are important for transport, and that the conformation of the three tightly coupled helices may play a critical role in sulphate transport. We also show that SHST1 undergoes a post-translational modification that is required for trafficking to the plasma membrane.

scholarly journals Functional identification of potential non-canonical N-glycosylation sites within Cav3.2 T-type calcium channels

2020 ◽  
Vol 13 (1) ◽  
Author(s):  
Vendula Ficelova ◽  
Ivana A. Souza ◽  
Leos Cmarko ◽  
Maria A. Gandini ◽  
Robin N. Stringer ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Calcium Channels ◽  
Low Voltage ◽  
Consensus Sequence ◽  
Site Directed Mutagenesis ◽  
Post Translational Modification ◽  
Functional Identification ◽  
Electrophysiological Recordings ◽  
Nervous System Function ◽  
And Function

Abstract Low-voltage-activated T-type calcium channels are important contributors to nervous system function. Post-translational modification of these channels has emerged as an important mechanism to control channel activity. Previous studies have documented the importance of asparagine (N)-linked glycosylation and identified several asparagine residues within the canonical consensus sequence N-X-S/T that is essential for the expression and function of Cav3.2 channels. Here, we explored the functional role of non-canonical N-glycosylation motifs in the conformation N-X-C based on site directed mutagenesis. Using a combination of electrophysiological recordings and surface biotinylation assays, we show that asparagines N345 and N1780 located in the motifs NVC and NPC, respectively, are essential for the expression of the human Cav3.2 channel in the plasma membrane. Therefore, these newly identified asparagine residues within non-canonical motifs add to those previously reported in canonical sites and suggest that N-glycosylation of Cav3.2 may also occur at non-canonical motifs to control expression of the channel in the plasma membrane. It is also the first study to report the functional importance of non-canonical N-glycosylation motifs in an ion channel.

scholarly journals MRTF transcription and Ezrin-dependent plasma membrane blebbing are required for entotic invasion

2017 ◽  
Vol 216 (10) ◽  
pp. 3087-3095 ◽  
Author(s):  
Laura Soto Hinojosa ◽  
Manuel Holst ◽  
Christian Baarlink ◽  
Robert Grosse
Keyword(s):  
Plasma Membrane ◽  
Cell Invasion ◽  
Serum Response Factor ◽  
Critical Role ◽  
Actin Dynamics ◽  
Actin Cortex ◽  
Tightly Coupled ◽  
Related Transcription Factor ◽  
Nuclear Shuttling ◽  
The Impact

Entosis is a nonapoptotic form of cell death initiated by actomyosin-dependent homotypic cell-in-cell invasion that can be observed in malignant exudates during tumor progression. We previously demonstrated formin-mediated actin dynamics at the rear of the invading cell as well as nonapoptotic plasma membrane (PM) blebbing in this cellular motile process. Although the contractile actin cortex involved in bleb-driven motility is well characterized, a role for transcriptional regulation in this process has not been studied. Here, we explore the impact of the actin-controlled MRTF–SRF (myocardin-related transcription factor–serum response factor) pathway for sustained PM blebbing and entotic invasion. We find that cortical blebbing is tightly coupled to MRTF nuclear shuttling to promote the SRF transcriptional activity required for entosis. Furthermore, PM blebbing triggered SRF-mediated up-regulation of the metastasis-associated ERM protein Ezrin. Notably, Ezrin is sufficient and important to sustain bleb dynamics for cell-in-cell invasion when SRF is suppressed. Our results highlight the critical role of the actin-regulated MRTF transcriptional pathway for bleb-associated invasive motility, such as during entosis.

scholarly journals Cysteine residues in the Na+/dicarboxylate co-transporter, NaDC-1

1999 ◽  
Vol 344 (1) ◽  
pp. 205-209 ◽  
Author(s):  
Ana M. PAJOR ◽  
Sally J. KRAJEWSKI ◽  
Nina SUN ◽  
Rama GANGULA
Keyword(s):  
Protein Trafficking ◽  
Direct Relationship ◽  
Transmembrane Domain ◽  
Cysteine Residue ◽  
Site Directed Mutagenesis ◽  
Cysteine Residues ◽  
Directed Mutagenesis ◽  
Transport Activity ◽  
Specific Reagent

The role of cysteine residues in the Na+/dicarboxylate co-transporter (NaDC-1) was tested using site-directed mutagenesis. The transport activity of NaDC-1 was not affected by mutagenesis of any of the 11 cysteine residues, indicating that no individual cysteine residue is necessary for function. NaDC-1 is sensitive to inhibition by the impermeant cysteine-specific reagent, p-chloromercuribenzenesulphonate (pCMBS). The pCMBS-sensitive residues in NaDC-1 are Cys-227, found in transmembrane domain 5, and Cys-476, located in transmembrane domain 9. Although cysteine residues are not required for function in NaDC-1, their presence appears to be important for protein stability or trafficking to the plasma membrane. There was a direct relationship between the number of cysteine residues, regardless of location, and the transport activity and expression of NaDC-1. The results indicate that mutagenesis of multiple cysteine residues in NaDC-1 may alter the shape or configuration of the protein, leading to alterations in protein trafficking or stability.

scholarly journals Exposure of Phosphatidylserine by Xk-related Protein Family Members during Apoptosis

2014 ◽  
Vol 289 (44) ◽  
pp. 30257-30267 ◽  
Author(s):  
Jun Suzuki ◽  
Eiichi Imanishi ◽  
Shigekazu Nagata
Keyword(s):  
Plasma Membrane ◽  
Amino Acid ◽  
Cell Surface ◽  
Family Members ◽  
Site Directed Mutagenesis ◽  
Pcr Analysis ◽  
Directed Mutagenesis ◽  
Amino Acid Residues ◽  
Related Protein ◽  
Transmembrane Regions

Apoptotic cells expose phosphatidylserine (PtdSer) on their surface as an “eat me” signal. Mammalian Xk-related (Xkr) protein 8, which is predicted to contain six transmembrane regions, and its Caenorhabditis elegans homolog CED-8 promote apoptotic PtdSer exposure. The mouse and human Xkr families consist of eight and nine members, respectively. Here, we found that mouse Xkr family members, with the exception of Xkr2, are localized to the plasma membrane. When Xkr8-deficient cells, which do not expose PtdSer during apoptosis, were transformed by Xkr family members, the transformants expressing Xkr4, Xkr8, or Xkr9 responded to apoptotic stimuli by exposing cell surface PtdSer and were efficiently engulfed by macrophages. Like Xkr8, Xkr4 and Xkr9 were found to possess a caspase recognition site in the C-terminal region and to require its direct cleavage by caspases for their function. Site-directed mutagenesis of the amino acid residues conserved among CED-8, Xkr4, Xkr8, and Xkr9 identified several essential residues in the second transmembrane and second cytoplasmic regions. Real time PCR analysis indicated that unlike Xkr8, which is ubiquitously expressed, Xkr4 and Xkr9 expression is tissue-specific.

Mutational analysis of transmembrane histidines in the amiloride-sensitive Na+/H+ exchanger

1995 ◽  
Vol 269 (2) ◽  
pp. C392-C402 ◽  
Author(s):  
D. Wang ◽  
D. F. Balkovetz ◽  
D. G. Warnock
Keyword(s):  
Mutational Analysis ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Transport Activity ◽  
Wild Type ◽  
Clear Cut ◽  
Inhibition Concentration ◽  
Nhe1 Activity ◽  
Per Se ◽  
Nhe Isoforms

The histidine-reactive reagent, diethyl pyrocarbonate (DEPC) inhibits the human amiloride-sensitive Na+/H+ exchanger (NHE1) in stably transfected fibroblasts. NHE1 was protected by cimetidine and amiloride from DEPC, and DEPC inhibition was reversed with hydroxylamine, suggesting a role for critical histidine groups in NHE activity. We replaced the histidines (H) in putative transmembrane domains (H35, H120, H349) with glycine (G) using site-directed mutagenesis. There was no significant change in NHE activity of the H120G; H349G; H120,349G; and H35,120,349G mutants compared with wild type. The 50% inhibition concentration values for amiloride, ethyl isopropyl amiloride (EIPA), and cimetidine of the H349G mutant were significantly increased compared with the wild-type NHE1. We also examined the DEPC effect on the transport activity of the triple histidine mutant (H35,120,349G) and found that NHE1 activity was still inhibited by DEPC with reversal by hydroxylamine and protected by amiloride and cimetidine. Kinetic analysis of DEPC inhibition indicated that two "critical" histidine residues are required for NHE transport activity. Substitutions of H349 with asparagine (N), glutamine (Q), serine (S), tyrosine (Y), valine (V), leucine (L), and phenylalanine (F) were also examined. There were no changes in NHE activity of these mutants compared with wild type. The H349G and H349L mutants became more resistant to amiloride, whereas the H349Y and H349F mutants became more sensitive to amiloride. The H349S (mimics NHE3) and H349Y (mimics NHE4) mutations had only modest effects on amiloride sensitivity. These results indicate that H349 affects the interaction of NHE1 with its inhibitors, even though substitutions at this site, per se, do not appear to explain the differences in amiloride sensitivity between different NHE isoforms. Despite clear-cut effects of the H349G mutation on the competitive interaction of NHE1 with cimetidine and EIPA, this mutation did not affect the affinity of NHE1 for its cationic substrates (Na+, Li+).

Hexose/H+ symporters in lower and higher plants.

1994 ◽  
Vol 196 (1) ◽  
pp. 483-491
Author(s):  
T Caspari ◽  
A Will ◽  
M Opekarová ◽  
N Sauer ◽  
W Tanner
Keyword(s):  
Higher Plants ◽  
Site Directed Mutagenesis ◽  
Hexose Transporter ◽  
Transmembrane Helices ◽  
Transport Activity ◽  
Large Families ◽  
Polymerase Chain ◽  
Selection For ◽  
Made In

A well-studied transporter of plant cells is the hexose/H+ symporter of the unicellular alga Chlorella kessleri. Its properties, studied in vivo, are briefly summarized. In part, they are atypical and it has been suggested that this porter acts in an asymmetric way. Three genes coding for Chlorella hexose transport activity have been identified (HUP1, HUP2 and HUP3). HUP1 cDNA expressed in a mutant of Schizosaccharomyces pombe not transporting any D-glucose has been studied in detail. Several mutants with changed Km values for substrate were obtained, some by random polymerase chain reaction mutation and selection for decreased sensitivity towards the toxic sugar 2-deoxyglucose, some by site-directed mutagenesis. The amino acids affected clustered in the centre of the putative transmembrane helices V, VII and XI. Large families of hexose transporter genes are found in higher plants (Arabidopsis, Chenopodium, Ricinus). Their functional role is discussed. Finally, the progress made in studying plant transporters in a vesicle system energized by cytochrome c oxidase is summarized.

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