Isolation and characterization of cDNA clones encoding proteins interacted with Rac1Vr of mung bean using yeast two-hybrid system

Abstract

The Rho small GTP-binding proteins of the Ras superfamily consist of Rho, Rac, and Cdc42 subfamiles in animal cells and yeasts. Plants have a unique subfamily of Rho GTPase, termed Rop (Rho-related GTPase from plant) in addition to Rac subfamily, and Rops and Racs may act as a common switch in signaling to many aspects of plant growth, development, and defense responses through the distinct effectors, which activate specific downstream signaling events. In this study, the yeast two-hybrid system was employed to identify proteins that interact with Rac1Vr protein of mung bean. As an expression cDNA library in yeast, cDNA library of mung bean hypocotyl was constructed. The library contains 2 x 10^6 independent clones, has at least 98% of recombinants and 1.1 kb of average insert size. From the screening of the library with Rac1Vr as a bait, 342 positive candidates were obtained. Forty candidates out of 342 positive candidates were further analyzed and divided into 11 distinct groups. One group has homology with Ribosomal-associated p40 protein that play a role in protein translation. This fact suggests that Rac1Vr is associated with translational machinery and is involved in growth and development by controlling of translational rate. The second group is a clathrin heavy chain. Because the growing cells are highly secretory and require a high level of plasma membrane recycling to maintain the architecture, Rac1Vr might be involved in endocytotic trafficking by clathrin-dependent manner. The third group has serine/threoine kinase domain that is possible to be a receptor-like serine/threoine kinase (RLKs) in plants suggesting that Rac1Vr also receive the signals through interacting with RLKs directly like other Rops. All these interactions might be involved in signaling of morphology and development in plant. ; Ras superfamily에 속하는 Rho GTPase 는 동물과 yeast에서 그 구조와 기능에 따라 Rac, Rho, Cdc42로 구분되어진다. 식물에서는 이것과 구분되는 Rop (Rho-related GTPase from Plant) 이라는 독특한 Rho GTPase family 가 존재하며, Rop은 특정한 신호전달 과정을 활성화하는 다양한 effector들과의 상호작용을 통해 식물의 성장과 발달과정, 방어기작 등에 관여하는 것으로 보고되고 있다. 본 논문에서는 식물에서의 Rho GTPase의 신호전달 과정을 이해하기 위해, yeast two-hybrid system을 이용하여 녹두의 Rac1Vr과 interaction하는 단백질을 암호화하는 유전자를 분리, 동정하였다. Yeast에서 발현되는 cDNA library로서, 총 2 x 10^6 개의 클론 중 98% 이상이 재조합 클론이고, 평균 1.1 kb의 insert크기를 가지는 library를 제작하였다. Rac1Vr로 이 cDNA library를 screening한 결과, 총 342개의 positive candidate를 얻었고, 이 중 40개의 candidate들에 대한 제한효소 분석을 통해 각기 다른 11개의 그룹으로 나누어 염기서열 분석을 수행한 후, database 검색을 통해 상동성을 가진 유전자를 조사해 봄으로써 이 유전자들의 기능을 추론하였다. 11개의 candidate 중에서, ribosomal-associated p40 protein, clathrin heavy chain, 그리고 serine/threonine kinase가 동정 되었고, 이 단백질들과 Rac1Vr과의 interaction은 아마도 일반적인 식물의 형태와 발달과정에서의 신호전달에 관여하는 것으로 생각된다.