College of Life Sciences, Shaanxi Normal University, Xi'an 710119, China.
Int J Mol Sci. 2022 May 22;23(10):5811. doi: 10.3390/ijms23105811.
Redundancy and lethality is a long-standing problem in genetics but generating minimal and lethal phenotypes in the knockouts of the same gene by different approaches drives this problem to a new high. In Asn (N)-linked glycosylation, a complex and ubiquitous cotranslational and post-translational protein modification required for the transfer of correctly folded proteins and endoplasmic reticulum-associated degradation (ERAD) of misfolded proteins, ALG12 (EBS4) is an α 1, 6-mannosyltransferase catalyzing a mannose into GlcManGlcNAc. In , T-DNA knockout is lethal while likely null mutants isolated by forward genetics are most healthy as weak alleles, perplexing researchers and demanding further investigations. Here, we isolated a true null allele, , with the W258Stop mutation in ALG12/EBS4. restored the sensitivity of brassinosteroid receptor mutants , , and with ER-trapped BRI1 to brassinosteroids. Furthermore, maturated earlier than the wild-type. Moreover, concomitant with impaired and misfolded proteins accumulated in the ER, had higher sensitivity to tunicamycin and salt than the wild-type. Our findings thus clarify the role of SBI2/ALG12/EBS4 in the regulation of the ERAD of misfolded glycoproteins, and plant growth and stress response. Further, our study advocates the necessity and importance of using multiple approaches to validate genetics study.
冗余和致死性是遗传学中长期存在的问题，但通过不同方法对同一基因的敲除产生最小和致死表型，将这一问题推向了新的高度。在天冬酰胺（N）-连接糖基化中，ALG12（EBS4）是一种 α1,6-甘露糖基转移酶，催化将甘露糖转移到 GlcManGlcNAc 中，是一种复杂且普遍的共翻译和翻译后蛋白质修饰，对于正确折叠的蛋白质的转移和错误折叠的蛋白质的内质网相关降解（ERAD）是必需的。在，T-DNA 敲除是致死的，而通过正向遗传学分离的可能的 null 突变体作为弱等位基因是最健康的，这让研究人员感到困惑，并要求进一步的研究。在这里，我们分离出一个真正的 null 等位基因，W258Stop 突变在 ALG12/EBS4 中。恢复了 brassinosteroid 受体突变体，，和 ER 捕获的 BRI1 对 brassinosteroids 的敏感性。此外，成熟得比野生型早。此外，伴随着内质网中积累的受损和错误折叠的蛋白质，对衣霉素和盐的敏感性比野生型更高。我们的研究结果因此阐明了 SBI2/ALG12/EBS4 在调节错误折叠糖蛋白、植物生长和应激反应的 ERAD 中的作用。此外，我们的研究提倡使用多种方法来验证遗传学研究的必要性和重要性。
