CENP-A chaperone Scm3HJURP contains a cysteine-rich domain required for centromere targeting and kinetochore integrity. CENP-A chaperone Scm3HJURP contains a cysteine-rich domain required for centromere targeting and kinetochore integrity

Centromeric chromatin is crucial for supporting kinetochore assembly and chromosome segregation. To specify centromeres, the histone H3 variant CENP-A is loaded by the conserved chaperone Scm3/HJURP. The amino-terminus of Scm3/HJURP interacts with CENP-A, while the carboxyl-terminus contains a small domain that interacts with the Mis18 holocomplex, which facilitates centromere localization. Fungal Scm3 proteins contain an additional conserved cysteine-rich domain (CYS) with unknown function. In this study, we found that CYS is essential for the localization and function of fission yeast Scm3. Disrupting CYS prevents Scm3 from localizing to the centromere and results in cell lethality. Moreover, introducing point mutations in a predicted zinc-binding motif within CYS hinders Scm3 centromere targeting and kinetochore integrity, consistent with structural evidence that CYS binds zinc. We find that expressing only the Scm3 carboxyl-terminus is harmful as it localizes to centromeres and causes defective kinetochores. Remarkably, mutating the CYS zinc-binding motif in this construct eliminates the observed centromere localization and toxicity. Together, our findings establish this cysteine-rich domain with predicted zinc-binding capability as an essential feature of fungal Scm3 proteins and shed new light on the molecular mechanism for targeting Scm3 to centromeres. Overall design: Chromatin immunoprecipitation DNA-sequencing (ChIP-seq) for CENP-A chaperone Scm3, as well as a Scm3-∆CYS mutants and untagged control in fission yeast Schizosaccharomyces pombe.

着丝粒染色质对于支持动粒组装与染色体分离至关重要。为了特化着丝粒，组蛋白H3变体CENP-A由保守的组蛋白分子伴侣Scm3/HJURP负责装载。Scm3/HJURP的氨基末端可与CENP-A相互作用，而其羧基末端则包含一个可与Mis18全复合物结合的小型结构域，该结构域能够促进Scm3的着丝粒定位。真菌来源的Scm3蛋白还额外含有一个功能未知的保守半胱氨酸富集结构域（CYS）。本研究发现，CYS结构域对于裂殖酵母Scm3的定位与功能至关重要。破坏CYS结构域会阻止Scm3定位于着丝粒，并导致细胞致死。此外，在CYS结构域内的预测锌结合基序中引入点突变，会阻碍Scm3的着丝粒靶向过程并破坏动粒完整性，这与CYS可结合锌的结构生物学证据相一致。我们还发现，仅表达Scm3羧基末端会产生毒性：该片段可定位于着丝粒并引发动粒功能异常。值得注意的是，在此截短片段中突变CYS的锌结合基序，则可消除其着丝粒定位与毒性表型。综上，本研究证实这种具有预测锌结合能力的半胱氨酸富集结构域是真菌Scm3蛋白的必需元件，并为Scm3靶向着丝粒的分子机制提供了新的见解。整体实验设计：对粟酒裂殖酵母（Schizosaccharomyces pombe）中的CENP-A分子伴侣Scm3、Scm3-ΔCYS突变体以及未标记的对照组样本进行染色质免疫沉淀测序（ChIP-seq）。