To identify the zinc transport function of the membrane proteins Gt1 and Zrt1 in Komagataella phaffii (Pichia pastoris) and study their regulatory mode. Two membrane proteins that might have zinc transport function were found in K. phaffii. GT1 was known to encode a glycerol transporter belonging to the Major Facilitator Superfamily. ZRT1 was predicted to resemble the zinc transporter gene in Saccharomyces cerevisiae. Consistent with the prediction, protein plasma-membrane localizations were confirmed by ultracentrifugation and confocal microscopy. Their zinc binding abilities were identified by ITC in vitro, and the impaired zinc uptake activity caused by their deficiencies was confirmed by zinc fluorescence quantification in vivo. Furthermore, zinc excess could turn the two channels off, while zinc deficiency induced their expressions. Gt1 could only function to maintain zinc homeostasis in glycerol, while the block of Gt1 function might lead to Zrt1 upregulation in glucose. The zinc transport capabilities of Gt1 and Zrt1 were identified in vivo and in vitro. Their regulatory mode to maintain zinc homeostasis in K. phaffii is a new inspiration.

中文翻译：

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甘油转运蛋白 1 (Gt1) 和锌调节转运蛋白 1 (Zrt1) 以不同模式在 Komagataella phaffii（毕赤酵母）中发挥锌稳态作用

鉴定Komagataella phaffii (Pichia pastoris)中膜蛋白Gt1和Zrt1的锌转运功能并研究其调控模式。在 K. phaffii 中发现了两种可能具有锌转运功能的膜蛋白。已知 GT1 编码属于主要促进剂超家族的甘油转运蛋白。预计 ZRT1 类似于酿酒酵母中的锌转运蛋白基因。与预测一致，通过超速离心和共聚焦显微镜证实了蛋白质质膜定位。它们的锌结合能力在体外由 ITC 鉴定，并且由它们的缺陷引起的锌吸收活性受损通过体内锌荧光定量得到证实。此外，锌过量会关闭这两个通道，而锌缺乏会导致它们的表达。Gt1 只能维持甘油中的锌稳态，而 Gt1 功能的阻断可能导致葡萄糖中 Zrt1 的上调。Gt1 和 Zrt1 的锌转运能力在体内和体外得到鉴定。他们在 K. phaffii 中维持锌稳态的调节模式是一个新的灵感。