Abstract
The C terminus of the rat brain Na(+)-Ca(2+) exchanger (RBE-1; NCX1. 4) (amino acids 875-903) is modeled to contain the last transmembrane alpha helix (amino acids 875-894) and an intracellular extramembraneous tail of 9 amino acids (895-903). Truncation of the last 9 C-terminal amino acids, Glu-895 to stop, did not significantly impair functional expression in HeLa or HEK 293 cells. Truncation, however, of 10 amino acids (Leu-894 to stop; mutant C10) reduced Na(+) gradient-dependent Ca(2+) uptake to 35-39% relative to the wild type parent exchanger, and further truncation of 13 or more amino acids resulted in expression of trace amounts of transport activity. Western analysis indicated that Na(+)-Ca(2+) exchanger protein was produced whether transfection was carried out with functional or non-functional mutants. Immunofluorescence studies of HEK 293 cells expressing N-Flag epitope-tagged wild type and mutant Na(+)-Ca(2+) exchangers revealed that transport activity in whole cells correlated with surface expression. All cells expressing the wild type exchanger or C9 exhibited surface expression of the protein. Only 39% of the cells expressing C10 exhibited surface expression, and none was detected in cells transfected with non-functional mutants C13 and C29. Since functional and non-functional mutants were glycosylated, the C terminus is not mandatory to translocation into the endoplasmic reticulum (ER). Endoglycosidase H digestion of [(35)S]methionine-labeled protein derived from wild type Na(+)-Ca(2+) exchanger and from C10 indicated that resistance to the digestion was acquired after 1 and 5 h of chase, respectively. C29 did not acquire detectable resistance to endoglycosidase H digestion even after 10 h of chase. Taken together, these results suggest that the "cellular quality control machinery" can tolerate the structural change introduced by truncation of the C terminus up to Ser-893 albeit with reduced rate of ER-->Golgi transfer and reduced surface expression of the truncated protein. Further truncation of C-terminal amino acids leads to retention of the truncated protein in the ER, no transfer to the Golgi, and no surface expression.
Highlights
Hydropathy analysis of the cloned NCX1 (Naϩ-Ca2ϩ exchanger) gene indicated that the protein can be organized into 12 transmembrane helices, the first of which was suggested to be a cleavable signal peptide [2]
We have shown that the signal peptide truncated protein is glycosylated in vitro when dog pancreatic microsomes are added to a reticulocyte lysate expression system [4], suggesting that the nascent protein translocates into the lumen of endoplasmic reticulum (ER)2 in the absence of its signal peptide
Based on hydropathy analysis [14], the profile-fed neural network system [15], and the recently published revised topological models based on scanning cysteine accessibility method [16, 17], the 31 C-terminal amino acids (Table I) of the NCX1 gene are modeled to contain the last transmembrane ␣ helix [10] and an extramembraneous tail
Summary
Construction of C-terminal Truncated Mutants—The method of Kunkel [7] was used to prepare C-terminal truncated mutants of the rat brain clone rbe-1 [8]. 48 h after transfection, intracellular Naϩ was raised in the cells by 10-min incubation at 37 °C with 0.16 M NaCl, 0.02 M Tris-HCl, pH 7.4, 0.02 M MgCl2, 25 M nystatin, and 1 mM ouabain. The Naϩ-preloaded cells were collected by centrifugation, and suspended in a minimal volume of the Naϩ preloading solution (without nystatin and MgCl2,) Part of these cells was used to determine Naϩ gradient-dependent Ca2ϩ uptake, part for protein determination, and part for Western analysis. Reconstitution of Transfected Cell Proteins—Cells expressing the wild type and C-terminal truncated mutants of the Naϩ-Ca2ϩ exchanger were reconstituted into exogenous brain phospholipids. Cellular Localization of the Naϩ-Ca2ϩ Exchanger—Indirect immunofluorescence of N-terminal Flag-tagged wild type and mutated NaϩCa2ϩ exchangers expressed in HEK 293 cells was carried out as described for HeLa cells in Ref. 10. The image analysis of the confocal images including pseudo color representation, brightness, and contrast level were carried out using the standard Zeiss software package and the Adobe Photoshop 4.0 program (Adobe Systems, Inc., Mountain View, CA)
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