Merwin Jason R, Bogar Lucien B, Poggi Sarah B, Fitch Rebecca M, Johnson Arlen W, Lycan Deborah E
Biochemistry and Molecular Biology Program, Lewis & Clark College, Portland, Oregon 97219.
Department of Molecular Biosciences, University of Texas, Austin, Texas 78712.
Genetics. 2014 Nov;198(3):1071-85. doi: 10.1534/genetics.114.168294. Epub 2014 Sep 10.
Ribosome biogenesis has been studied extensively in the yeast Saccharomyces cerevisiae. Yeast Ltv1 is a conserved 40S-associated biogenesis factor that has been proposed to function in small subunit nuclear export. Here we show that Ltv1 has a canonical leucine-rich nuclear export signal (NES) at its extreme C terminus that is both necessary for Crm1 interaction and Ltv1 export. The C terminus of Ltv1 can substitute for the NES in the 60S-export adapter Nmd3, demonstrating that it is a functional NES. Overexpression of an Ltv1 lacking its NES (Ltv1∆C13) was strongly dominant negative and resulted in the nuclear accumulation of RpS3-GFP; however, export of the pre-40S was not affected. In addition, expression of endogenous levels of Ltv1∆C protein complemented both the slow-growth phenotype and the 40S biogenesis defect of an ltv1 deletion mutant. Thus, if Ltv1 is a nuclear export adapter for the pre-40S subunit, its function must be fully redundant with additional export factors. The dominant negative phenotype of Ltv1∆NES overexpression was suppressed by co-overexpressing RpS3 and its chaperone, Yar1, or by deletion of the RpS3-binding site in Ltv1∆NES, suggesting that titration of RpS3 by Ltv1∆NES is deleterious in yeast. The dominant-negative phenotype did not correlate with a decrease in 40S levels but rather with a reduction in the polysome-to-monosome ratio, indicating reduced rates of translation. We suggest that titration of RpS3 by excess nuclear Ltv1 interferes with 40S function or with a nonribosomal function of RpS3.
核糖体生物合成已在酿酒酵母中得到广泛研究。酵母Ltv1是一种保守的与40S相关的生物合成因子，有人提出它在小亚基核输出中发挥作用。在这里，我们表明Ltv1在其极端C末端具有一个典型的富含亮氨酸的核输出信号（NES），这对于Crm1相互作用和Ltv1输出都是必需的。Ltv1的C末端可以替代60S输出衔接子Nmd3中的NES，表明它是一个功能性NES。缺乏NES的Ltv1（Ltv1∆C13）的过表达具有强烈的显性负性作用，并导致RpS3-GFP在细胞核中积累；然而，前40S的输出不受影响。此外，内源性水平的Ltv1∆C蛋白的表达补充了ltv1缺失突变体的生长缓慢表型和40S生物合成缺陷。因此，如果Ltv1是前40S亚基的核输出衔接子，其功能必须与其他输出因子完全冗余。通过共过表达RpS3及其伴侣Yar1，或通过缺失Ltv1∆NES中的RpS3结合位点，可抑制Ltv1∆NES过表达的显性负性表型，这表明Ltv1∆NES对RpS3的滴定在酵母中是有害的。显性负性表型与40S水平的降低无关，而是与多核糖体与单核糖体比率的降低有关，表明翻译速率降低。我们认为，过量的核Ltv1对RpS3的滴定会干扰40S功能或RpS3的非核糖体功能。
