Role of Cysteine Residues in the N-Terminal Extracellular Domain of the Human VIP 1 Receptor for Ligand Binding A Site-directed Mutagenesis Studya

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.

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The role of cysteine residues 129 and 329 in Escherichia coli K1 CMP-NeuAc synthase

Biochemical Journal ◽

10.1042/bj2950485 ◽

1993 ◽

Vol 295 (2) ◽

pp. 485-491 ◽

Cited By ~ 4

Author(s):

G Zapata ◽

P P Roller ◽

J Crowley ◽

W F Vann

Keyword(s):

Escherichia Coli ◽

Specific Activity ◽

Site Directed Mutagenesis ◽

Cysteine Residues ◽

Directed Mutagenesis ◽

Wild Type ◽

Acetylneuraminic Acid ◽

Denatured States ◽

Escherichia Coli K1

N-Acetylneuraminic acid cytidyltransferase (CMP-NeuAc synthase) of Escherichia coli K1 is sensitive to mercurials and has cysteine residues only at positions 129 and 329. The role of these residues in the catalytic activity and structure of the protein has been investigated by site-directed mutagenesis and chemical modification. The enzyme is inactivated by the thiol-specific reagent dithiodipyridine. Inactivation by this reagent is decreased in the presence of the nucleotide substrate CTP, suggesting that a thiol residue is at or near the active site. Site-directed mutagenesis of either residue Cys-129 to serine or Cys-329 to selected amino acids has minor effects on the specific activity of the enzyme, suggesting that cysteine is not essential for catalysis and that a disulphide bond is not an essential structural component. The limited reactivity of the enzyme to other thiol-blocking reagents suggests that its cysteine residues are partially exposed. The accessibility and role of the cysteine residues in enzyme structure were investigated by fluorescence, c.d. and denaturation studies of wild-type and mutant enzymes. The mutation of Cys-129 to serine makes the enzyme more sensitive to heat and chemical denaturation, but does not cause gross changes in the protein structure as judged by the c.d. spectrum. The mutant containing Ser-129 instead of Cys-129 had a complex denaturation pathway similar to that of wild-type E. coli K1 CMP-NeuAc synthase consisting of several partially denatured states. Cys-329 reacts more readily with N-[14C]ethylmaleimide when the enzyme is in a heat-induced relaxed state. Cys-129 is less reactive and is probably a buried residue.

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The role of cysteine residues of spinach ferredoxin-NADP+ reductase as assessed by site-directed mutagenesis

Biochemistry ◽

10.1021/bi00076a010 ◽

1993 ◽

Vol 32 (25) ◽

pp. 6374-6380 ◽

Cited By ~ 49

Author(s):

Alessandro Aliverti ◽

Luciano Piubelli ◽

Giuliana Zanetti ◽

Thomas Luebberstedt ◽

Reinhold G. Herrmann ◽

...

Keyword(s):

Site Directed Mutagenesis ◽

Cysteine Residues ◽

Directed Mutagenesis

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Role of post-translational modifications at the β-subunit ectodomain in complex association with a promiscuous plant P4-ATPase

Biochemical Journal ◽

10.1042/bcj20160207 ◽

2016 ◽

Vol 473 (11) ◽

pp. 1605-1615 ◽

Cited By ~ 11

Author(s):

Sara R. Costa ◽

Magdalena Marek ◽

Kristian B. Axelsen ◽

Lisa Theorin ◽

Thomas G. Pomorski ◽

...

Keyword(s):

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Β Subunit ◽

Post Translational Modifications ◽

Functional Roles ◽

Evolutionarily Conserved ◽

A Site ◽

P Type ◽

Cell Division Control

P-type ATPases of subfamily IV (P4-ATPases) constitute a major group of phospholipid flippases that form heteromeric complexes with members of the Cdc50 (cell division control 50) protein family. Some P4-ATPases interact specifically with only one β-subunit isoform, whereas others are promiscuous and can interact with several isoforms. In the present study, we used a site-directed mutagenesis approach to assess the role of post-translational modifications at the plant ALIS5 β-subunit ectodomain in the functionality of the promiscuous plant P4-ATPase ALA2. We identified two N-glycosylated residues, Asn181 and Asn231. Whereas mutation of Asn231 seems to have a small effect on P4-ATPase complex formation, mutation of evolutionarily conserved Asn181 disrupts interaction between the two subunits. Of the four cysteine residues located in the ALIS5 ectodomain, mutation of Cys86 and Cys107 compromises complex association, but the mutant β-subunits still promote complex trafficking and activity to some extent. In contrast, disruption of a conserved disulfide bond between Cys158 and Cys172 has no effect on the P4-ATPase complex. Our results demonstrate that post-translational modifications in the β-subunit have different functional roles in different organisms, which may be related to the promiscuity of the P4-ATPase.

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