Sanmartín Maite, Ordóñez Angel, Sohn Eun Ju, Robert Stephanie, Sánchez-Serrano José Juán, Surpin Marci A, Raikhel Natasha V, Rojo Enrique
Departamento de Genética Molecular de Plantas, Centro Nacional de Biotecnología, Consejo Superior de Investigaciones Científicas, E-28049 Madrid, Spain.
Proc Natl Acad Sci U S A. 2007 Feb 27;104(9):3645-50. doi: 10.1073/pnas.0611147104. Epub 2007 Feb 20.
The protein storage vacuole (PSV) is a plant-specific organelle that accumulates reserve proteins, one of the main agricultural products obtained from crops. Despite the importance of this process, the cellular machinery required for transport and accumulation of storage proteins remains largely unknown. Interfering with transport to PSVs has been shown to result in secretion of cargo. Therefore, secretion of a suitable marker could be used as an assay to identify mutants in this pathway. CLV3, a negative regulator of shoot stem cell proliferation, is an extracellular ligand that is rendered inactive when targeted to vacuoles. We devised an assay where trafficking mutants secrete engineered vacuolar CLV3 and show reduced meristems, a phenotype easily detected by visual inspection of plants. We tested this scheme in plants expressing VAC2, a fusion of CLV3 to the vacuolar sorting signal from the storage protein barley lectin. In this way, we determined that trafficking of VAC2 requires the SNARE VTI12 but not its close homologue, the conditionally redundant VTI11 protein. Furthermore, a vti12 mutant is specifically altered in transport of storage proteins, whereas a vti11 mutant is affected in transport of a lytic vacuole marker. These results demonstrate the specialization of VTI12 and VTI11 in mediating trafficking to storage and lytic vacuoles, respectively. Moreover, they validate the VAC2 secretion assay as a simple method to isolate genes that mediate trafficking to the PSV.
蛋白质储存液泡（PSV）是植物特有的细胞器，它积累储备蛋白，储备蛋白是从作物中获得的主要农产品之一。尽管这一过程很重要，但储存蛋白运输和积累所需的细胞机制仍 largely 未知。干扰向PSV的运输已被证明会导致货物分泌。因此，分泌一种合适的标记物可以用作鉴定该途径中突变体的测定方法。CLV3是茎干细胞增殖的负调控因子，是一种细胞外配体，当靶向液泡时会失活。我们设计了一种测定方法，其中运输突变体分泌工程化的液泡CLV3，并显示分生组织减少，这种表型通过对植物的目视检查很容易检测到。我们在表达VAC2的植物中测试了该方案，VAC2是CLV3与储存蛋白大麦凝集素的液泡分选信号的融合体。通过这种方式，我们确定VAC2的运输需要SNARE蛋白VTI12，而不需要其密切同源物、条件性冗余的VTI11蛋白。此外，vti12突变体在储存蛋白运输方面有特异性改变，而vti11突变体在溶酶体液泡标记物的运输方面受到影响。这些结果证明了VTI12和VTI11分别在介导向储存液泡和溶酶体液泡的运输中的特异性。此外，它们验证了VAC2分泌测定法是分离介导向PSV运输的基因的一种简单方法。
