Abstract
The rat brain Na(+)-Ca2+ exchanger (RBE) gene, as well as other isoforms of this protein family, can be organized into 12 transmembrane alpha helices, the first of which was proposed by Durkin et al. (14) to constitute a cleavable signal peptide. We have prepared three amino-terminal mutants, in which 21, 26, and 31 amino acids beyond the initiating methionine were deleted. The deletions include the hydrophobic core of the putative signal peptide (N21), the entire putative signal peptide and parts of the putative signal peptidase cleavage site (N26), and the entire putative signal peptide and putative signal peptidase cleavage site (N31). All three mutant clones were transiently expressed in HeLa cells. The average Na+ gradient-dependent Ca2+ transport activity of the mutant exchangers was 108% (N21), 37.2% (N26), and 60.06% (N31) of the wild-type clone. Mutation of the putative cleavage site by an exchange of Ala-32 --> Asp, resulted in a decrease in Na(+)-Ca2+ exchange activity to 7.7%, relative to the wild-type exchanger. Functional reconstitution of the proteins that were expressed in the transfected cells, resulted in transport activities of: 60.1% (N21), 26.75% (N26), 85.36% (N31), and 31% (Ala-32 --> Asp) relative to the wild-type exchanger. Western blot analysis of the protein profile of RBE-1, N21, N26, N31 and Ala-32 --> Asp-transfected HeLa cells was carried out by using an antipeptide antibody directed against a pentadecapeptide segment derived from the large putative cytoplasmic loop of the cloned rat exchanger gene. In the total cell extract and in the plasma membrane-enriched fraction, in addition to a major protein band of about 125 kDa, which corresponds to the molecular mass of the mature fully processed Na(+)-Ca2+ exchanger, an additional protein of about 135 kDa is revealed in the profile of N21- and N26-transfected cells. This band is not detected in the protein profile of RBE-1, N31, or Ala-32 -->Asp. The amino-terminal truncated mutants of the cloned Na(+)-Ca2+ exchanger could be expressed and processed also in a reticulocyte lysate supplemented with dog microsomes. Our results suggest that the putative signal peptide of the cloned Na(+)-Ca2+ exchanger gene does not play a mandatory role in functional expression of the protein in HeLa cells.
Highlights
The nucleotide sequencers) reported in this paper has been submitted to the GenBankTM / EMBL Data Bank with accession numberts) X68812
We have used either a [a- 32P]dCTP-labeled polymerase chain reaction-amplified DNA fragment of 863 base pairs corresponding to nucleotides 1936-2799 of the cloned exchanger gene or a [y- 32P]ATP -labeled 53-mer synthetic antisense oligonucleotide corresponding to nucleotides 14-67 of the following sequence: 5'-AGAGAGCCACCAGAGTTACCAGACGAAATCCCATTGAAACATTGGGTGGGAGAC-3'
The Role of the Amino-terminal Segment of the Cloned Na +Ca2 + Exchanger Gene in Functional Expression-In order to investigate the role of the amino-terminal segment of the cloned exchanger gene rat brain Na+_Ca2+ exchanger (RBE)-l in functional expression of Na +Ca 2+ exchange activity, we have prepared three amino-terminal deletion mutants of the protein: N21, N26, and N31
Summary
Preparation of RBE-l1 Mutants-Truncation of clone RBE-l [9] has been carried out by the method of Kunkel [15]. We have used either a [a- 32P]dCTP-labeled polymerase chain reaction-amplified DNA fragment of 863 base pairs corresponding to nucleotides 1936-2799 of the cloned exchanger gene or a [y- 32P]ATP -labeled 53-mer synthetic antisense oligonucleotide corresponding to nucleotides 14-67 (the numbering refers to the open reading frame of the cloned exchanger RBE-1) of the following sequence: 5'-AGAGAGCCACCAGAGTTACCAGACGAAATCCCATTGAAACATTGGGTGGGAGAC-3'. The second part was pelleted, suspended in a minimal volume of phosphate-buffered saline, clarified by a 10-s sonication, and used without further treatment This total cell extract contained the entire repertoire of proteins produced in the transfected HeLa cells. For Western blots, the antiserum was purified by preadsorption on HeLa cells This was done by incubating the antiserum with the cells at a dilution of 1:20 (in 3% bovine serum albumin, 0.02% NaN3 in phosphate-buffered saline) for 60 min at 25 DC, after which the cells were sedimented and discarded. In the P30 fraction, the specific activity of the enzyme increased between 3- and 6-fold in different preparations over the total cell extract and about 15-fold over the P100 fraction
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