Functional analysis of SpHMA1 N-terminus in yeast: regulation of cadmium (Cd) transport by his-rich motifs

Abstract

Cadmium (Cd) poses significant toxicity risks to most biological systems. In the hyperaccumulator Sedum plumbizincicola, the HMA1 plays a key role in Cd detoxification through chloroplast–mediated process. Our previous work demonstrated that SpHMA1 actively exports Cd from chloroplasts, thereby protecting photosynthesis by reducing Cd accumulation within these organelles. In this study, we identified a Cd–selective functional domain in SpHMA1 and characterizes two histidine–rich motifs within its N–terminal region. Through heterologous expression of full–length SpHMA1 and its N–terminal truncation mutants in yeast, we systematically analyzed their functional roles in heavy metal transport. Under Cd stress, SpHMA1Δ2–46–expressing cells showed altered growth patterns compared to full–length SpHMA1 transformants, despite comparable total Cd accumulation. Notably, truncation of the first 80 amino acids (SpHMA1Δ2–80) significantly increased metal sensitivity (Cd, Zn, Cu) in Δycf1 yeast, accompanied by a significant increase in intracellular Cd accumulation. Complete deletion of the histidine–rich domain (SpHMA1Δ2–107) restored metal tolerance to empty vector–control levels. Our findings demonstrate functional differentiation between the dual his–rich motifs in heavy metal transport regulation, advancing understanding of HMA1′s molecular mechanisms in plant metal homeostasis.