Nuclear localization signal of murine CMP-Neu5Ac synthetase includes residues required for both nuclear targeting and enzymatic activity.
5-N-Acetylneuraminic acid (Neu5Ac) is the major sialic acid derivative found in animal cells. As a component of cell surface glycoconjugates, Neu5Ac is pivotal to numerous cellular recognition and communication processes including host-parasite interactions. A prerequisite for the synthesis of sialylated glycoconjugates is the activation of Neu5Ac to cytidine-monophosphate N-acetylneuraminic acid (CMP-Neu5Ac). The reaction is catalyzed by CMP-Neu5Ac-synthetase (syn), which, for unknown reasons, resides in the nucleus. Sequence analysis of the cloned murine CMP-Neu5Ac synthetase identified three clusters of basic amino acids (BC1-BC3) that might function as nuclear localization signals (NLS). In the present study chimeric protein and mutagenesis strategies were used to show that BC1 and BC2 are active NLS sequences when attached to the green fluorescent protein (enhanced GFP), but only BC2 is necessary and sufficient to mediate the nuclear import of CMP-Neu5Ac synthetase. Site-directed mutations identified the residues K(198)RXR to be essential for nuclear transport and Arg(202) to be necessary to complete the transport process. Cytoplasmic forms of CMP-Neu5Ac synthetase generated by single site mutations in BC2 demonstrated that (i) enzyme activity is independent of nuclear localization, and (ii) Arg(199) and Arg(202) are involved in both nuclear transport and synthetase activity. Comparison of all known and predicted CMP-sialic acid synthetases reveals Arg(202) and Gln(203) as highly conserved in evolution and critically important for optimal synthetase activity but not for nuclear localization. Combined, the data demonstrate that nuclear transport and enzyme activity are independent functions that share some common amino acid requirements in CMP-Neu5Ac synthetase.
Munster
Anja-K
AK
Institut für Physiologische Chemie/Proteinstruktur, Medizinische Hochschule Hannover, Carl-Neuberg-Strasse 1, 30625 Hannover, Germany.
Weinhold
Birgit
B
Gotza
Birgit
B
Muhlenhoff
Martina
M
Frosch
Matthias
M
Gerardy-Schahn
Rita
R
D016475:3T3 Cells; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry; D000818:Animals; D001120:Arginine / Q000737:chemistry; D001483:Base Sequence; D015153:Blotting, Western; D016466:CHO Cells; D002467:Cell Nucleus / Q000201:enzymology* / Q000378:metabolism; D017124:Conserved Sequence; D006224:Cricetinae; D003568:Cytidine Monophosphate / Q000378:metabolism*; D003593:Cytoplasm / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D019143:Evolution, Molecular; D005973:Glutamine / Q000737:chemistry; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000378:metabolism; D051379:Mice; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D000216:N-Acylneuraminate Cytidylyltransferase / Q000737:chemistry* / Q000378:metabolism*; D019913:Nuclear Localization Signals; D010957:Plasmids / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D000596:Amino Acids; D008164:Luminescent Proteins; D019913:Nuclear Localization Signals; D011993:Recombinant Fusion Proteins; D005973:Glutamine; D049452:Green Fluorescent Proteins; D001120:Arginine; D000216:N-Acylneuraminate Cytidylyltransferase; D003568:Cytidine Monophosphate