Heme is the major source of iron and an important component of respiratory enzyme. However, excessive level of free heme is cytotoxic and specific trafficking pathway is necessary for heme delivery to its target site. While the synthesis of heme had been well characterized, the pathway for heme trafficking remain poorly understood due to lacking structural information of such heme transporter. As an effort to uncover these pathway, studies have been conducted on Caenorhabditis elegans, a heme auxotroph that solely dependent on dietary heme for survival. Following that, a gene of novel heme transporter known as heme-responsive gene-4 (HRG-4) was recently discovered. The aim of this study is to elucidate the molecular mechanism of heme transport by HRG-4 as a model of heme transporter in animals.
Recombinant HRG-4 from C. elegans (CeHRG-4) was overexpressed in Pichia pastoris, a methylotrophic yeast. The protein solubilization and purification was tested over a range of detergents. The oligomerization state varies with the change in detergent. Further optimization step will be conducted to improve the purification quality. In vivo yeast spot assay was conducted to study the role of CeHRG-4 in hemin and PPIX transport. CeHRG-4 was able to transport hemin efficiently. On the other hand, a relatively low cell growth was observed on plates supplemented with PPIX. Based on the observation from yeast spot assay, spectroscopic study was conducted to analyze hemin and PPIX binding affinity to CeHRG-4. CeHRG-4 showed a weak binding to hemin and no binding affinity was detected with PPIX. These structural and functional properties will be discussed in detail during the presentation.