[논문]Kinesin superfamily KIF1A와 결합하는 미세소관 불안정화 단백질 SCG10의 규명

문일수; 석대현

doi:10.5352/jls.2009.19.7.859

Kinesin superfamily KIF1A와 결합하는 미세소관 불안정화 단백질 SCG10의 규명
SCG10, a Microtubule-Destabilizing Factor, Interacts Directly with Kinesin Superfamily KIF1A Protein in Brain 원문보기

생명과학회지 = Journal of life science, v.19 no.7 = no.111, 2009년, pp.859 - 865

문일수 (동국대학교 의과대학 해부학교실) , 석대현 (인제대학교 의과대학 생화학교실)

초록
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미세소관은 세포골격단백질의 중요한 구성 단백질로 축삭돌기 내에서는 세포막 방향으로 정렬되어 있다. Kinesin superfamily (KIFs)는 세포 내에서 미세소관을 따라 세포 내 소포들을 운반하는 분자 자동차 (molecular motor) 단백질이다. 본 연구에서 우리는 효모 two-hybrid system을 사용하여 KIF1A의 coiled-coil 영역과 결합하는 단백질로 미세소관 불안정화 요소인 SCG10 단백질을 분리하였다. SCG10은 KIFs에서 KIF1A와만 특이적으로 결한 하며, KIF1A의 400에서 820아미노산 부위가 SCG10과의 결합에 필수적임을 효모 two-hybrid assay로 확인하였다. 또한 SCG10의 coiled-coil영역은 KIF1A와의 결합에 필수영역임을 확인하였으며 단백질간의 결합은 Glutathione S-transferase pull-down assay를 통하여 확인하였다. 생쥐의 뇌 파쇄액에 SCG10항체로 면역침강을 행하여 KIF1A를 확인한 결과KIF1A는 SCG10과 특이적으로 같이 침강하였다. 이러한 결과들은 KIF1A는 SCG10와 결합하여 SCG10이 포함된 소포를 미세소관을 따라 이동시킴을 시사한다.

Abstract ▼ AI-Helper

Microtubules, a major cytoskeleton, form parallel arrays in the axon and are oriented with their plus ends toward the cell periphery. Kinesin superfamily proteins (KIFs) are the molecular motors acting in the microtubule-based motilities of organelles in cells. Here, we used the yeast two-hybrid system to identify the protein that interacts with the coiled-coil domain of KIF1A and found a specific interaction with microtubule-destabilizing factor SCG10. SCG10 bound to the amino acid residues between 400 and 820 of KIF1A, but not to other KIFs in the yeast two-hybrid assay. The coiled-coil domain of SCG10 is essential for interaction with KIF1A. In addition, this specific interaction was also observed in the Glutathione S-transferase pull-down assay. An antibody to SCG10 specifically co-immunoprecipitated KIF1A associated with SCG10 from mouse brain extracts. These results suggest that KIF1A motor protein transports SCG10-containing vesicles along microtubules in neurons.

주제어

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가설 설정

aa, the amino acid residue number. (C) Schematic representation of the SCG10 truncation clones. Several truncated forms of SCG10 were tested in the yeast two-hybrid assay for interaction with KIF1A, +, interaction with KIF1A; no interaction with KIF1A.

제안 방법

in neurons. In this work, we have identified SCG10 is KIFlA-binding "receptor" in KIFlA-dependent anterograde transport cargoes, thus revealing a new sorting/transporting system for transport of cargo vesicle in neurons.
We did not analyze further the rest eight clones which encoded unknown proteins. To identify the region of KIF1A required for the interaction with SCG10, we constructed deletion mutants of KIF1A and analyzed their interactions with SCG10 using the yeast two-hybrid assay (Fig. IB). This experiment demonstrated that the minimal binding domain was located in a small region of KIF1A corresponding to amino acids 400-820.
SCG10 contains three functional domains: membrane anchoring (MA), regulatory, and coiled-coil domains [15, 31]. To identify the region of SCG10 required for the interaction with KIF1A, we constructed deletion mutants of SCG10 and analyzed their interactions with KIF1A using the yeast two-hybrid assay. MA domain and regulatory domain did not bind KIF1A.

대상 데이터

1A). The three positive SCG10 clones (clones 2, 3 and 6) overlapped at the open reading frame (ORF) of SCG10 (Fig. 1A). We did not analyze further the rest eight clones which encoded unknown proteins.

이론/모형

Library inserts were then amplified by PCR and analyzed by restriction enzyme digestion. Unique inserts were sequenced, and DNA and protein sequence analysis were performed with the BLAST algorithm at the National Center for Biotechnology Information (NCBI). After isolation of the plasmids encoding the library clones, these plasmids were tested far interactions of the reporter gene in yeast by the retransformation.

성능/효과

To clarify whether SCG10 interacts specifically with KIF1A or whether the interaction includes other isoforms of KIF1A, the tails of KIF1A, KIF1B, KIF1C, KIF3B, KIF5B and KIF17 were tested for binding with SCG10 (Fig. 2). There was no detectable binding between SCG10 and the tail domains of the other major neuronal KIF, such as KIF17.

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