The Amino-Terminus of Nitric Oxide Sensitive Guanylyl Cyclase α1 Does Not Affect Dimerization but Influences Subcellular Localization

Background: Nitric oxide sensitive guanylyl cyclase (NOsGC) is a heterodimeric enzyme formed by an alpha- and a beta(1)-subunit. A splice variant (C-alpha(1)) of the alpha(1)-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 alpha(1)-subunit are mandatory for quantitative heterodimerization implying that the C-alpha(1)-splice variant should lose its capacity to dimerize quantitatively. Methodology/Principal Findings: In the current study we demonstrate preserved quantitative dimerization of the C-alpha(1)-splice by co-purification with the beta(1)-subunit. In addition we used fluorescence resonance energy transfer (FRET) based on fluorescence lifetime imaging (FLIM) using fusion proteins of the beta(1)-subunit and the alpha(1)-subunit or the C-alpha(1) variant with ECFP or EYFP. Analysis of the respective combinations in HEK-293 cells showed that the fluorescence lifetime was significantly shorter (approximate to 0.3 ns) for alpha(1)/beta(1) and C-alpha(1)/beta(1) than the negative control. In addition we show that lack of the amino-terminus in the alpha(1) splice variant directs it to a more oxidized subcellular compartment. Significance: We conclude that the amino-terminus of the alpha(1)-subunit is dispensable for dimerization in-vivo and ex-vivo, but influences the subcellular trafficking.