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- Title
The Amino-Terminus of Nitric Oxide Sensitive Guanylyl Cyclase a1 Does Not Affect Dimerization but Influences Subcellular Localization.
- Authors
Kraehling, Jan R.; Busker, Mareike; Haase, Tobias; Haase, Nadine; Koglin, Markus; Linnenbaum, Monika; Behrends, Soenke
- Abstract
Background: Nitric oxide sensitive guanylyl cyclase (NOsGC) is a heterodimeric enzyme formed by an a- and a b1-subunit. A splice variant (C-a1) of the a1-subunit, lacking at least the first 236 amino acids has been described by Sharina et al. 2008 and has been shown to be expressed in differentiating human embryonic cells. Wagner et al. 2005 have shown that the amino acids 61-128 of the a1-subunit are mandatory for quantitative heterodimerization implying that the C-a1-splice variant should lose its capacity to dimerize quantitatively. Methodology/Principal Findings: In the current study we demonstrate preserved quantitative dimerization of the C-a1- splice by co-purification with the b1-subunit. In addition we used fluorescence resonance energy transfer (FRET) based on fluorescence lifetime imaging (FLIM) using fusion proteins of the b1-subunit and the a1-subunit or the C-a1 variant with ECFP or EYFP. Analysis of the respective combinations in HEK-293 cells showed that the fluorescence lifetime was significantly shorter (<0.3 ns) for a1/b1 and C-a1/b1 than the negative control. In addition we show that lack of the aminoterminus in the a1 splice variant directs it to a more oxidized subcellular compartment. Conclusions/Significance: We conclude that the amino-terminus of the a1-subunit is dispensable for dimerization in-vivo and ex-vivo, but influences the subcellular trafficking.
- Subjects
NITRIC oxide; GUANYLATE cyclase; RADIOACTIVITY; AMINO acids; ENERGY transfer; TOXICOLOGY; FLUORESCENCE
- Publication
PLoS ONE, 2011, Vol 6, Issue 9, p1
- ISSN
1932-6203
- Publication type
Article
- DOI
10.1371/journal.pone.0025772