Fission Yeast인 Schizosaccharomyces pombe의 Kinesin-related Protein1 단백질의 특성 : Characterization of the Kinesin- related Protein 1 in Fission Yeast, Schizosaccharomyces pombe
- 발행기관 고려대학교
- 발행년도 2005
- 학위수여년월 2005. 8
- 학위명 박사
- 학과 대학원 생명공학과 분자생물학전공
- 식별자(기타) DL:000015976576
- 서지제어번호 000045225171
초록/요약
Kinesin은 세포성장과 분열에 관련된 다양한 기능을 가진 microtubule-based motor protein이다. 모든 kinesin은 필수 구성분인 motor domain과 cargo가 결합하는 variable region을 가지고 있다. Kinesin은 ATP로부터 방출된 화학 에너지를 기계적인 에너지로 바꾸어 이동하게 된다. Kinesin과 kinesin-related proteins (KRPs)은 다양한 생물종에서 막결합 세포소기관과 vesicle의 이동에 관여하는 것으로 알려져 있다. 또한 KRPs는 세포분열의 여러 단계에서 spindle 구조와 기능, microtubule dynamics, chromatin 구조와 기능, 염색체의 이동, 세포질 분열 및 세포 극성에 중요한 역할을 하는 것으로 알려져 있다. 새로운 kinesin 유사 유전자를 분리하기 위하여 여러 생물종의 kinesin motor domain간에 유사성이 매우 높은 ATP 결합부위와 microtubule 결합부위를 이용하여 primer를 합성하였다. 이 primer를 이용하여 S. pombe의 genomic DNA를 주형으로 증폭하여 해당유전자의 일부를 얻었다. 이렇게 얻어진 유전자 단편의 일부를 probe로 이용하여 S. pombe library에서 plaque and colony hybridization을 수행하였고, 그 결과 새로운 유전자인 krp1+ (kinesin-related protein 1)을 클로닝하였다. krp1+은 554 amino acids로 구성된 61.9 kDa인 단백질을 암호화하는 1,665 bps의 ORF를 가졌으며, KHC (kinesin heavy chain) superfamily에 속한다는 것을 알 수 있었다 (GeneBank accession No. AF156966 (genomic DNA) and AF247188 (mRNA)). Krp1p의 역할을 연구하기 위하여, S. pombe에서 krp1+ 유전자를 파쇄 혹은 과량발현 하였다. krp1+ null allele을 포함하는 균주는 생존하였고, Krp1p가 과량발현된 균주는 비정상적으로 길어지며 생존하지 못하였다. Krp1p는 cytoplasmic microtubules에 위치하며, late interphase에 세포의 양 끝에 모이는 것을 알 수 있었다. Krp1p의 보다 자세한 세포 내 기능을 연구하기 위하여, 두 군데의 ATP-binding motifs를 site-directed mutagenesis하거나, leucine zipper-like motif(LZiP)를 truncation하였으며, 정상적인 Krp1p와 세가지 다른 돌연변이 형태들의 Krp1p (ATP1p, ATP2p, ΔLZiPp)을 세포 내에서 각각 과량발현 하였다. Krp1p를 과량발현한 균주에서 hyper-extended microtubules과 비정상적인 핵의 모양이 관찰되었다. 또한, Krp1p의 ATP-binding domain 1 (G89E)에 돌연변이가 일어난 균주는 ATP1p가 과량발현 되어도 야생형과 흡사한 모습을 나타내었다. 반면에 ATP-binding domain 2 (G238E)에 돌연변이가 일어난 균주는 ATP2p가 과량발현 되었을 때, cell polarity가 변경되었다. 그리고, Krp1p C-terminal에 위치한 leucine zipper-like domain (LZiP)을 truncation한 균주는 ΔLZiPp가 과량발현 되었을 때, 비정상적으로 길어지며 hyper-extended spindle을 가지나, 정상적인 핵분열을 수행하는 것을 알 수 있었다. 위와 같은 결과들을 종합해 볼 때, Krp1p는 fission yeast인 S. pombe에서 microtubule polymerization과 cell-polarized growth, 그리고 핵의 모양 유지에 관여한다는 것을 알 수 있었다.
more초록/요약
Kinesins are microtubule-based motor proteins with various functions related to the cell growth and division. Kinesins convert chemical energy released from nucleoside triphosphates, preferentially from ATP, into mechanical energy. The activities of a variety of molecular motors are required to spatially organize microtubules and chromosomes in the mitotic spindle. Some motors contribute to spindle pole focusing, others stabilize antiparallel microtubule overlaps in the spindle midzone, and yet others are involved in chromosome-microtubule interactions. In this study, a kinesin-related protein, named Krp1p, was identified in Schizosaccharomyces pombe and characterized. The primer sequences were driven from the highly conserved area of the kinesin genes in other organisms, and then a kinesin gene was cloned from S. pombe using PCR technique. Sequence analysis reveals that krp1+ has a 1,665 bp open reading frame (ORF) encoding a protein consisted of 554 amino acids with a molecular weight of 61.9 kDa and is homologous to the proteins that belong to the kinesin heavy chain (KHC) superfamily (GeneBank accession No. AF156966 (genomic DNA) and AF247188 (mRNA). To characterize the Krp1p, the gene was disrupted in S. pombe, but the null mutant was viable. When Krp1p fused to GFP was overexpressed in fission yeast, Krp1p is localized on microtubules. To investigate the roles of motifs in Krp1p, three different mutant forms of Krp1p in fission yeast were constructed by site-directed mutagenesis in two ATP-binding motifs or by truncation of the leucine zipper-like motif (LZiP) and the mutated krp1 gene was overexpressed. In the intact Krp1p-overexpressed fission yeast, hyper-extended microtubules and the aberrant nuclear shape were observed. As a functional consequence, a point mutation of ATP-binding domain 1 (G89E) in Krp1p reversed the effect of Krp1p overexpression in fission yeast, whereas the specific mutation in ATP-binding domain 2 (G238E) resulted in the altered cell polarity. Additionally, truncation of the leucine zipper-like domain (LZiP) at the C-terminal of Krp1p showed a normal nuclear division. Taken together, it can be suggested that krp1p could be involved in regulation of cell-polarized growth through ATP-binding motifs in fission yeast.
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Table of contents
Table of contents
List of Tables
List of Figures
Abbreviations
Abstract
Introduction
Materials and Methods
1. Strains, cell culture, and plasmids
2. PCR amplifications and isolation of PCR products
3. DNA manipulations and hybridization
4. Isolation and cloning of the krp1+ gene
5. Construction of krp1::ura4+ null mutant
6. Fluorescence and phase contrast microscopy
7. Site-directed mutagenesis and modification of krp1+
8. Antibody production of Krp1p
9. Immunoblot analysis and affinity purification
Results
1. Identification of the genes encoding kinesin-related proteins in Schizosaccharomyces pombe
2. Cloning and sequencing analysis of Krp1+
3. Similarity of Krp1+ to KHC kinesin superfamily
4. Deletion of Krp1+
5. Preparation of Krp1p-specific Antibodies and Identification of the Krp1p
6. Morphological changes of Schizosaccharomyces pombe by Krp1p overexpression
7. Krp1p overexpression induced hyper-extended microtubules
8. Cellular localization of GFP-Krp1p on cytoplasmic microtubules
9. The specific mutations in ATP-binding motifs altered cell morphology
10. Subcellular localization of mutagenized Krp1p in ATP binding motifs
11. The truncation of leucine zipper-like motif
Discussion
References
Abstract in Korean
List of Tables
TABLE 1. The kinesin-related proteins (KRPs) in S. pombe
TABLE 2. Yeast strains used in this study
TABLE 3. Media used in the culture of S. pombe
TABLE 4. Modification of Krp1p
List of Figures
Figure 1. Kinesin is a plus-end-directed microtubule motor protein
Figure 2. Kinesin tree
Figure 3. Model for microtubule regulation of cell polarity in fission yeast
Figure 4. A role for Klp5 and Klp6
Figure 5. Amino acid sequence comparison of the kinesin proteins and position of primers
Figure 6. PCR primer sequence for cloning of KRPs
Figure 7. Nucleotide sequence of the krp1+ gene region
Figure 8. Sequence alignment of Krp1p with other kinesin heavy chain (KHC) proteins
Figure 9. Percent of homology among KHC proteins
Figure 10. Phylogenetic tree of the KHC and KRP85/95 family kinesins showing that Krp1p is grouped with the KHC family
Figure 11. Construction of krp1::ura4+
Figure 12. Deletion of the krp1+ gene
Figure 13. Growth curve of krp1+ deletion mutant and wild type cells
Figure 14. Cellular effects of overexpressed Krp1p
Figure 15. Phase contrast microscopy of C-1, DK101 and DK102
Figure 16. The effects of middle level overexpressed Krp1p on microtubules
Figure 17. The effects of high level overexpressed Krp1p on microtubules
Figure 18. Immunoblot analysis for Krp1p and GFP-Krp1p using monoclonal anti-Krp1p antibody or anti-GFP antibody
Figure 19. Localization of Krp1p
Figure 20. Co-localization of Krp1p and microtubules
Figure 21. Schematic representation of modification in Krp1p
Figure 22. The molecular effect of G89E to ATP binding to the motor domain
Figure 23. Immunoblot analysis for Krp1p and its derivatives using monoclonal anti-Krp1p antibody
Figure 24. Alteration in viability of overexpression of mutated ATP-binding motifs in Krp1p
Figure 25. Alteration of cellular effect of overexpression of mutated ATP-binding motifs in Krp1p
Figure 26. Localization of Krp1p with the mutation in ATP-binding motifs
Figure 27. The effects of overexpressed Krp1p with the muta- tion in ATP-binding motifs on microtubules
Figure 28. Immunoblot analysis for Krp1p and its derivatives using monoclonal anti-Krp1p antibody or anti-GFP antibody
Figure 29. Alteration in viability of overexpression of Krp1p with the truncated leucine zipper-like motif (LZiP)
Figure 30. Localization and effect of Krp1p with the truncated leucine zipper-like motif (LZiP)
