Schizosaccharomyces pombe의 septin 유전자의 클로닝과 염기서열분석

김성철,김형배,Kim, Seong-chul,Kim, Hyoog Bai 한국미생물학회 1997 미생물학회지 Vol.33 No.4

Septin 유전자는 Saccharomyces cerevisiae에서 filament를 암호화하고 있으며 세포질분열이나 bud의 형성에 중요한 역할을 하는 것으로 알려져있다. S. cerevisiae에서 septin유전자는 4가지가 발견되었으며 초파리나 쥐의 세포에서도 발견되고 있다. 본 연구에서는 PCR 방법을 이용하여 Schizosaccharomyces pombe에서 septin 유전자를 찾아내었다. S. pombe의 septin 유전자는 1143 bp의 open reading frame을 갖고 있으며 380개의 아미노산으로된 42 kd의 분자량을 가진 단백질을 암호화하였다. S. cerevisiae의 septin 유전자의 하나인 $CDC_{12}$ 유전자와의 유사성을 비교한 결과 51.8%의 유사성이 있음이 밝혀졌다. It is known that septin gene encodes the filament in Saccharomyces cerevisiae and it has importants roles in bud formation and cytokinesis. Four septin genes have been cloned in S. cerevisiae and it was found in Drosophila melanogaster and mouse. In this study, we cloned the septin gene in Schizosaccahromyces pombe by use of PCR technique. The septin gene in S. pombe has an 1,143 bp open reading frame and encodes a protein of 380 amino acids with a molecular weight of 42 kd. Comparison of the predicted amano acid sequences between the septin gene in S. pombe and CDC12 gene in S. cerevisiae reveals the 51.8% of simility.

The correlation of Septin4 gene expression with sperm quality, DNA damage, and oxidative stress level in infertile patients

Rahil Jannatifar,Hamid Piroozmanesh,Fahimeh Naghi Jalalabadi,Hamid Reza Momeni 대한해부학회 2023 Anatomy & Cell Biology Vol.56 No.4

Septin4 belong to a family of polymerizing GTP-binding proteins that are required for many cellular functions, such as membrane compartmentalization, vesicular trafficking, mitosis, and cytoskeletal remodeling. Since, Septin4 is expressed specifically in the testis, we aimed to determine the association between Septin4 gene expression with sperm quality, DNA damage, and stress oxidative level in infertile patients. The present study included 60 semen samples that grouped into three groups: normozoospermia (n=20), asthenozoospermia (n=20), astheno-teratozoospermia (n=20). Initially, semen parameters were analyzed by using the World Health Organization protocol. The mRNA expression of Septin4 in sperm was examined using reverse transcription-polymerase chain reaction. Oxidative stress markers, i.e., total antioxidant capacity, superoxide dismutase, catalase, glutathione peroxidase, and malondialdehyde, were determined by ELISA kit. The current study showed a statistically significant highly positive correlation in Septin4 gene expression with sperm motility, normal morphology, viability, capacity, and sperm mitochondrial membrane potential (MMP). However, it showed significant negative correlation with sperm DNA fragmentation. Septin4 had a significant correlation with stress oxidative factor and antioxidant enzyme levels. In conclusion, Septin4 gene expression provides clinical useful information for the diagnosis of male infertility. It might be a marker for discrimination between fertile and infertile patients. The current study showed a statistically significant highly positive correlation in Septin4 gene expression with sperm motility, normal morphology, viability, capacity, and sperm MMP. However, it shows significant negative correlation with sperm DNA fragmentation. Septin4 had a significant correlation with stress oxidative factor and antioxidant enzyme levels.

Phosphorylation-Dependent Septin Interaction of Bni5 is Important for Cytokinesis

Sung Chang Nam,Hyeran Sung,Seung Hye Kang,Jin Young Joo,이수재,정연복,이종길,송석길 한국미생물학회 2007 The journal of microbiology Vol.45 No.3

In budding yeast, septin plays as a scaffold to recruits protein components and regulates crucial cellular events including bud site selection, bud morphogenesis, Cdc28 activation pathway, and cytokinesis. Phosphorylation of Bni5 isolated as a suppressor for septin defect is essential to Swe1-dependent regulation of bud morphogenesis and mitotic entry. The mechanism by which Bni5 regulates normal septin function is not completely understood. Here, we provide evidence that Bni5 phosphorylation is important for interaction with septin component Cdc11 and for timely delocalization from septin filament at late mitosis. Phosphorylation-deficient bni5-4A was synthetically lethal with hof1Δ . bni5-4A cells had defective structure of septin ring and connected cell morphology, indicative of defects in cytokinesis. Two-hybrid analysis revealed that bni5-4A has a defect in direct interaction with Cdc11 and Cdc12. GFP-tagged bni5-4A was normally localized at mother-bud neck of budded cells before middle of mitosis. In contrast, at large-budded telophase cells, bni5-4A-GFP was defective in localization and disappeared from the neck approximately 2 min earlier than that of wild type, as evidenced by time-lapse analysis. Therefore, earlier delocalization of bni5-4A from septin filament is consistent with phosphorylation-dependent interaction with the septin component. These results suggest that timely de localization of Bni5 by phosphorylation is important for septin function and regulation of cytokinesis. In budding yeast, septin plays as a scaffold to recruits protein components and regulates crucial cellular events including bud site selection, bud morphogenesis, Cdc28 activation pathway, and cytokinesis. Phosphorylation of Bni5 isolated as a suppressor for septin defect is essential to Swe1-dependent regulation of bud morphogenesis and mitotic entry. The mechanism by which Bni5 regulates normal septin function is not completely understood. Here, we provide evidence that Bni5 phosphorylation is important for interaction with septin component Cdc11 and for timely delocalization from septin filament at late mitosis. Phosphorylation-deficient bni5-4A was synthetically lethal with hof1Δ. bni5-4A cells had defective structure of septin ring and connected cell morphology, indicative of defects in cytokinesis. Two-hybrid analysis revealed that bni5-4A has a defect in direct interaction with Cdc11 and Cdc12. GFP-tagged bni5-4A was normally localized at mother-bud neck of budded cells before middle of mitosis. In contrast, at large-budded telophase cells, bni5-4A-GFP was defective in localization and disappeared from the neck approximately 2 min earlier than that of wild type, as evidenced by time-lapse analysis. Therefore, earlier delocalization of bni5-4A from septin filament is consistent with phosphorylation-dependent interaction with the septin component. These results suggest that timely delocalization of Bni5 by phosphorylation is important for septin function and regulation of cytokinesis.

Phosphorylation-Dependent Septin Interaction of Bni5 is Important for Cytokinesis

Nam, Sung-Chang,Sung, Hye-Ran,Kang, Seung-Hye,Joo, Jin-Young,Lee, Soo-Jae,Chung, Yeon-Bok,Lee, Chong-Kil,Song, Suk-Gil The Microbiological Society of Korea 2007 The journal of microbiology Vol.45 No.3

In budding yeast, septin plays as a scaffold to recruits protein components and regulates crucial cellular events including bud site selection, bud morphogenesis, Cdc28 activation pathway, and cytokinesis. Phosphorylation of Bni5 isolated as a suppressor for septin defect is essential to Swe1-dependent regulation of bud morphogenesis and mitotic entry. The mechanism by which Bni5 regulates normal septin function is not completely understood. Here, we provide evidence that Bni5 phosphorylation is important for interaction with septin component Cdc11 and for timely delocalization from septin filament at late mitosis. Phosphorylation-deficient bni5-4A was synthetically lethal with $hof1{\Delta}$. bni5-4A cells had defective structure of septin ring and connected cell morphology, indicative of defects in cytokinesis. Two-hybrid analysis revealed that bni5-4A has a defect in direct interaction with Cdc11 and Cdc12. GFP-tagged bni5-4A was normally localized at mother-bud neck of budded cells before middle of mitosis. In contrast, at large-budded telophase cells, bni5-4A-GFP was defective in localization and disappeared from the neck approximately 2 min earlier than that of wild type, as evidenced by time-lapse analysis. Therefore, earlier delocalization of bni5-4A from septin filament is consistent with phosphorylation-dependent interaction with the septin component. These results suggest that timely de localization of Bni5 by phosphorylation is important for septin function and regulation of cytokinesis.

Neurotoxicity and behavioral deficits associated with Septin 5 accumulation in dopaminergic neurons

Son, Jin H.,Kawamata, Hibiki,Yoo, Myung S.,Kim, Dae J.,Lee, Young K.,Kim, SooYoul,Dawson, Ted M.,Zhang, Hui,Sulzer, David,Yang, Lichuan,Beal, M. Flint,DeGiorgio, Lorraine A.,Chun, Hong S.,Baker, Harri Raven Press [etc.] 2005 Journal of neurochemistry Vol.94 No.4

<P>Abstract</P><P>Septin 5, a parkin substrate, is a vesicle- and membrane-associated protein that plays a significant role in inhibiting exocytosis. The regulatory function of Septin 5 in dopaminergic (DAergic) neurons of substantia nigra (SN), maintained at relatively low levels, has not yet been delineated. As loss of function mutations of parkin are the principal cause of a familial Parkinson's disease, a prevailing hypothesis is that the loss of parkin activity results in accumulation of Septin 5 which confers neuron-specific toxicity in SN-DAergic neurons. <I>In vitro</I> and <I>in vivo</I> models were used to support this hypothesis. In our well-characterized DAergic SN4741 cell model, acute accumulation of elevated levels of Septin 5, but not synphilin-1 (another parkin substrate), resulted in cytotoxic cell death that was markedly reduced by parkin co-transfection. A transgenic mouse model expressing a dominant negative parkin mutant accumulated moderate levels of Septin 5 in SN-DAergic neurons. These mice acquired a progressive <SMALL>L</SMALL>-DOPA responsive motor dysfunction that developed despite a 25% higher than normal level of striatal dopamine (DA) and no apparent loss of DAergic neurons. The phenotype of this animal, increased striatal dopamine and reduced motor function, was similar to that observed in parkin knockout animals, suggesting a common DAergic alteration. These data suggest that a threshold level of Septin 5 accumulation is required for DAergic cell loss and that <SMALL>L</SMALL>-DOPA-responsive motor deficits can occur even in the presence of elevated DA.</P>

Ankyrin Repeat-Rich Membrane Spanning/Kidins220 Protein Interacts with Mammalian Septin 5

Park, Han-Jeong,Park, Hwan-Woo,Lee, Shin-Jae,Arevalo, Juan Carlos,Park, Young-Seok,Lee, Seung-Pyo,Paik, Ki-Suk,Chao, Moses V.,Chang, Mi-Sook Korean Society for Molecular and Cellular Biology 2010 Molecules and cells Vol.30 No.2

Neurotrophin receptors utilize specific adaptor proteins to activate signaling pathways involved in various neuronal functions, such as neurite outgrowth and cytoskeletal remodeling. The Ankyrin-Repeat Rich Membrane Spanning (ARMS)/kinase D-interacting substrate-220 kDa (Kidins220) serves as a unique downstream adaptor protein of Trk receptor tyrosine kinases. To gain insight into the role of ARMS/Kidins220, a yeast two-hybrid screen of a rat dorsal root ganglion library was performed using the C-terminal region of ARMS/Kidins220 as bait. The screen identified a mammalian septin, Septin 5 (Sept5), as an interacting protein. Co-immunoprecipitation using lysates from transiently transfected HEK-293 cells revealed the specific interaction between ARMS/Kidins220 and Sept5. Endogenous ARMS/Kidins220 and Sept5 proteins were colocalized in primary hippocampal neurons and were also predominantly expressed at the plasma membrane and in the tips of growing neurites in nerve growth factor-treated PC12 cells. Mapping of Sept5 domains important for ARMS/Kidins220 binding revealed a highly conserved N-terminal region of Sept5. The direct interaction between ARMS/Kidins220 and Sept5 suggests a possible role of RMS/Kidins220 as a functional link between neurotrophin receptors and septins to mediate neurotrophin-induced intracellular signaling events, such as neurite outgrowth and cytoskeletal remodeling.

Ankyrin Repeat-Rich Membrane Spanning/Kidins220 Protein Interacts with Mammalian Septin 5

Han Jeong Park,박환우,이신재,Juan Carlos Arevalo,박영석,이승표,Ki-Suk Paik,Moses V. Chao,장미숙 한국분자세포생물학회 2010 Molecules and cells Vol.30 No.2

Neurotrophin receptors utilize specific adaptor proteins to activate signaling pathways involved in various neuronal functions, such as neurite outgrowth and cytoskeletal re-modeling. The Ankyrin-Repeat Rich Membrane Spanning (ARMS)/kinase D-interacting substrate-220 kDa (Kidins220) serves as a unique downstream adaptor protein of Trk receptor tyrosine kinases. To gain insight into the role of ARMS/Kidins220, a yeast two-hybrid screen of a rat dorsal root ganglion library was performed using the C-terminal region of ARMS/Kidins220 as bait. The screen identified a mammalian septin, Septin 5 (Sept5), as an interacting pro-tein. Co-immunoprecipitation using lysates from tran-siently transfected HEK-293 cells revealed the specific interaction between ARMS/Kidins220 and Sept5. Endoge-nous ARMS/Kidins220 and Sept5 proteins were co-localized in primary hippocampal neurons and were also predominantly expressed at the plasma membrane and in the tips of growing neurites in nerve growth factor-treated PC12 cells. Mapping of Sept5 domains important for ARMS/Kidins220 binding revealed a highly conserved N-terminal region of Sept5. The direct interaction between ARMS/Kidins220 and Sept5 suggests a possible role of ARMS/Kidins220 as a functional link between neurotrophin receptors and septins to mediate neurotrophin-induced intracellular signaling events, such as neurite outgrowth and cytoskeletal remodeling.

SEPT12 Interacts with SEPT6 and This Interaction Alters the Filament Structure of SEPT6 in Hela Cells

Ding, Xiangming,Yu, Wenbo,Liu, Ming,Shen, Suqin,Chen, Fang,Wan, Bo,Yu, Long Korean Society for Biochemistry and Molecular Biol 2007 Journal of biochemistry and molecular biology Vol.40 No.6

Septins are a family of conserved cytoskeletal GTPase forming heteropolymeric filamentous structure in interphase cells, however, the mechanism of assembly are largely unknown. Here we described the characterization of SEPT12, sharing closest homology to SEPT3 and SEPT9. It was revealed that subcelluar localization of SEPT12 varied at interphase and mitotic phase. While SEPT12 formed filamentous structures at interphase, it was localized to the central spindle and to midbody during anaphase and cytokinesis, respectively. In addition, we found that SEPT12 can interact with SEPT6 in vitro and in vivo, and this interaction was independent of the coiled coil domain of SEPT6. Further, co-expression of SEPT12 altered the filamentous structure of SEPT6 in Hela cells. Therefore, our result showed that the interaction between different septins may affect the septin filament structure.

GTP Binding Is Required for SEPT12 to Form Filaments and to Interact with SEPT11

Xiangming Ding,Wenbo Yu,Ming Liu,ShuQing Shen,Fang Chen,Lihuan Cao,Bo Wan,Long Yu 한국분자세포생물학회 2008 Molecules and cells Vol.25 No.3

Septins are a family of filament-forming GTP-binding proteins involved in a variety of cellular process such as cytokinesis, exocytosis, and membrane dynamics. Here we report the biochemical and immunocytochemical characterization of a recently identified mammalian septin, SEPT12. SEPT12 binds GTP in vitro, and a mutation (Gly56 to Asn) in the GTPbinding motif abolished binding. Immunocytochemical analysis revealed that wild-type SEPT12 formed filamentous structures when transiently expressed in Hela cells whereas SEPT12G56A generated large aggregates. In addition, wild-type SEPT12 failed to form filaments when coexpressed with SEPT12G56A. We also observed that GTP-binding by SEPT12 is required for interaction with SEPT11 but not with itself.

Septin 6 Regulates the Cytoarchitecture of Neurons through Localization at Dendritic Branch Points and Bases of Protrusions

Sun-Jung Cho,문일수,이현숙,Samikshan Dutta,송진영,Randall Walikonis 한국분자세포생물학회 2011 Molecules and cells Vol.32 No.1

Septins, a conserved family of GTP-binding proteins with a conserved role in cytokinesis, are present in eukaryotes ranging from yeast to mammals. Septins are also highly expressed in neurons, which are post-mitotic cells. Sep-tin6 (SEPT6) forms SEPT2/6/7 complexes in vivo. In this study, we produced a very specific SEPT6 antibody. Im-munocytochemisty (ICC) of dissociated hippocampal cultures revealed that SEPT6 was highly expressed in neurons. Developmentally, the expression of SEPT6 was very low until stage 3 (axonal outgrowth). Significant expression of SEPT6 began at stage 4 (outgrowth of dendrites). At this stage, SEPT6 clusters were positioned at the branch points of developing dendrites. In maturing and mature neurons (stage 5), SEPT6 clusters were positioned at the base of filopodia and spines, and pre-synaptic boutons. Detergent extraction experiments also indicated that SEPT6 is not a post-synaptic density (PSD) protein. Throughout morphologic development of neurons, SEPT6 always formed tiny rings (external diameter, ~0.5 m), which appear to be clusters at low magnification. When a Sept6 RNAi vector was introduced at the early developmental stage (DIV 2), a significant reduction in dendritic length and branch number was evident. Taken together, our results indicate that SEPT6 begins to be expressed at the stage of dendritic outgrowth and regulates the cytoarchitecture.