Os09g0494200 - Revision history

Zhchuny: /* Localization */

2014-06-03T15:51:46Z

‎Localization

Zhchuny: /* Localization */

2014-06-03T15:43:43Z

‎Localization

Zhchuny: /* Annotated Information */

2014-06-03T14:23:59Z

‎Annotated Information

Zhchuny: /* Labs working on this gene */

2014-06-03T14:15:04Z

‎Labs working on this gene

Zhchuny: /* Annotated Information */

2014-06-03T14:11:45Z

‎Annotated Information

Zhchuny: /* Structure */

2014-06-03T14:06:57Z

‎Structure

Zhchuny: /* Expression */

2014-06-03T13:34:23Z

‎Expression

Zhchuny: /* Annotated Information */

2014-06-03T13:33:47Z

‎Annotated Information

Zhchuny: /* References */

2014-06-03T13:31:35Z

‎References

Zhchuny: /* References */

2014-06-03T13:31:16Z

‎References

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 ===Localization===
-To determine whether BC15/OsCTL1 resides on the plasma membrane or in intracellular compartments, we further separated the total membrane fraction isolated from ''pBC15GFP'' transgenic plants into the plasma membrane and endomembrane fractions. Protein gel blotting showed that BC15/OsCTL1 is predominantly present in the cellular endomembrane systems (Fig. 4A). We then investigated whether BC15/OsCTL1 targets a particular subcellular organelle. Signal peptide (SP) sequences provide valuable clues regarding the subcellular localization of chitinases. In contrast to AtCTL1, which has a secretion SP at the N terminus (D score = 0.865 in the SP prediction by SignalP 4. 0), BC15/OsCTL1 lacks this motif (D score = 0 .2 77), suggesting that BC15/OsCTL1 and AtCTL1 have different subcellular localization patterns. We viewed the root epidermal and coleoptiles cells of plants expressing the ''pBC15GFP'' transgene, and the dot-like fluorescent signals were observed in those living cells (Fig. 4B). The dot-like signals were almost identical to those labeled by a Golgi marker, while we coexpressed CTL1-GFP and Man49-m Cherry in rice protoplast cells (Fig .4C). Therefore, OsCTL1 is localized in the Golgi apparatus. As a type II membrane protein, the C terminus of BC15/OsCTL1 that contains a catalytic domain is proposed to face the Golgi lumen. To verify this topological structure, we treated the membrane extracts from plants expressing ''BC15/OsCTL1-GFP'' with proteinase K in the presence or absence of the detergent Triton X-100. Immunoblotting assays revealed that GFP fused to the C terminus of BC15/OsCTL1 was sensitive to proteinase K treatment in the presence of Triton X-100 ( Fig. 4D ), indicating that the C terminus of BC15/OsCTL1 faces the Golgi lumen.
+To determine whether BC15/OsCTL1 resides on the plasma membrane or in intracellular compartments, we further separated the total membrane fraction isolated from ''pBC15GFP'' transgenic plants into the plasma membrane and endomembrane fractions. Protein gel blotting showed that BC15/OsCTL1 is predominantly present in the cellular endomembrane systems (Fig. 4A). We then investigated whether BC15/OsCTL1 targets a particular subcellular organelle. Signal peptide (SP) sequences provide valuable clues regarding the subcellular localization of chitinases. In contrast to AtCTL1, which has a secretion SP at the N terminus (D score = 0.865 in the SP prediction by SignalP 4. 0), BC15/OsCTL1 lacks this motif (D score = 0 .2 77), suggesting that BC15/OsCTL1 and AtCTL1 have different subcellular localization patterns. We viewed the root epidermal and coleoptiles cells of plants expressing the ''pBC15GFP'' transgene, and the dot-like fluorescent signals were observed in those living cells (Fig. 4B). The dot-like signals were almost identical to those labeled by a Golgi marker, while we coexpressed CTL1-GFP and Man49-m Cherry in rice protoplast cells (Fig .4C). Therefore, OsCTL1 is localized in the Golgi apparatus. As a type II membrane protein, the C terminus of BC15/OsCTL1 that contains a catalytic domain is proposed to face the Golgi lumen. To verify this topological structure, we treated the membrane extracts from plants expressing ''BC15/OsCTL1-GFP'' with proteinase K in the presence or absence of the detergent Triton X-100. Immunoblotting assays revealed that GFP fused to the C terminus of BC15/OsCTL1 was sensitive to proteinase K treatment in the presence of Triton X-100 (Fig. 4D), indicating that the C terminus of BC15/OsCTL1 faces the Golgi lumen.
 [[File:four.jpg]]

← Older revision		Revision as of 15:43, 3 June 2014
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	===Localization===		===Localization===
		+	To determine whether BC15/OsCTL1 resides on the plasma membrane or in intracellular compartments, we further separated the total membrane fraction isolated from ''pBC15GFP'' transgenic plants into the plasma membrane and endomembrane fractions. Protein gel blotting showed that BC15/OsCTL1 is predominantly present in the cellular endomembrane systems (Fig. 4A). We then investigated whether BC15/OsCTL1 targets a particular subcellular organelle. Signal peptide (SP) sequences provide valuable clues regarding the subcellular localization of chitinases. In contrast to AtCTL1, which has a secretion SP at the N terminus (D score = 0.865 in the SP prediction by SignalP 4. 0), BC15/OsCTL1 lacks this motif (D score = 0 .2 77), suggesting that BC15/OsCTL1 and AtCTL1 have different subcellular localization patterns. We viewed the root epidermal and coleoptiles cells of plants expressing the ''pBC15GFP'' transgene, and the dot-like fluorescent signals were observed in those living cells (Fig. 4B). The dot-like signals were almost identical to those labeled by a Golgi marker, while we coexpressed CTL1-GFP and Man49-m Cherry in rice protoplast cells (Fig .4C). Therefore, OsCTL1 is localized in the Golgi apparatus. As a type II membrane protein, the C terminus of BC15/OsCTL1 that contains a catalytic domain is proposed to face the Golgi lumen. To verify this topological structure, we treated the membrane extracts from plants expressing ''BC15/OsCTL1-GFP'' with proteinase K in the presence or absence of the detergent Triton X-100. Immunoblotting assays revealed that GFP fused to the C terminus of BC15/OsCTL1 was sensitive to proteinase K treatment in the presence of Triton X-100 ( Fig. 4D ), indicating that the C terminus of BC15/OsCTL1 faces the Golgi lumen.
		+
		+	[[File:four.jpg]]

	==Labs working on this gene==		==Labs working on this gene==

← Older revision		Revision as of 14:23, 3 June 2014
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	[[File:three.jpg]]		[[File:three.jpg]]
		+
		+	===Localization===

	==Labs working on this gene==		==Labs working on this gene==

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 ==Labs working on this gene==
-Please input related labs here.
+* Key Laboratory of Plant Genomics, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China
 ==References==

← Older revision		Revision as of 14:11, 3 June 2014
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	[[File:three.jpg]]		[[File:three.jpg]]
−
−	~~===Evolution===~~
−	~~Please input evolution information here.~~
−
−	~~You can also add sub-section(s) at will.~~

	==Labs working on this gene==		==Labs working on this gene==

← Older revision		Revision as of 14:06, 3 June 2014
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	===Structure===		===Structure===
		+	''BC15'' Encodes a Chitinase-Like Protein. The coding sequence of ''BC15'' (981 nucleotides) encodes a protein comprising 326 amino acids. Pfam analysis showed that BC15 belongs to the GH19 family (PF00182) and is a class II chitinase (EC 3.2.1.14). Plaza 2.5 software further identified 27 orthologs (ORTHO004207) and 306 homologs (HOM000272) of BC15 in different plant species. Because all of them are plant chitinases, ''BC15'' thus encodes a plant chitinase. To check whether BC15 preserves the catalytic and substrate-binding domains of chitinases, we aligned the sequences of ''BC15'' and its orthologs. BC15 possesses the conserved signatures of GH19. One common signature of GH19 proteins is devoid of the N-terminal Cys-rich region that is considered for chitinase binding. Moreover, the H-E-T-T motif required for catalytic activity is replaced by the S-K-T-S motif. Construction of a phylogenetic tree that includes all of the BC15 orthologs and its homologs from rice and Arabidopsis indicated that the closest ortholog of BC15 is AtCTLl. Therefore, BC15 was designated as ''Chitinase-Like Protein1'' (''OsCTL1'') and will henceforth be referred to as ''BC15/OsCTL1'' in this paper.
		+	----
		+	BC15/OsCTL1 is an N-Glycosylated Membrane Protein. Plant chitinases often target the extra cellular matrix or various organelles. To investigate the locations where in BC15/OsCTL1 exists, we used the TMHMM 2.0 and SignalP 4.0 software to predict whether BC15/OsCTL1 has any localization motifs. These analyses indicated that BC15/OsCTL1 is a classical type II membrane protein and possesses an N-terminal transmembrane domain (Fig. 3A). To verify this prediction in vivo, we generated transgenic bc15 mutant plants that express GFP-fused BC15/OsCTL1 (pBC15GFP; Fig. 2B). The resulting construct fully rescued the mutant phenotypes, indicating that the tagging with GFP does not affect BC15/OsCTL1 function. We extracted the total proteins from pBC15GFP transgenic plants, fractionated them into soluble, total membrane, and cell wall fractions, and probed them using anti-GFP antibody; BC15/OsCTL1 was mainly detected in the membrane fraction (Fig. 3B), suggesting that BC15/OsCTL1 is membrane associated. This result prompted us to treat the membrane fraction with several chemicals. As shown in Figure 3C, BC15/OsCTL1 was partially solubilized following Triton X-100 treatment. BC15/O sCTL1 is thus an integral membrane protein. In addition, the Prositescan algorithm predicts that BC15/OsCTL1 has three N-glycosylation sites. Changes in the extent of migration of BC15/CTL1-GFP on a SDS-PAGE gel after treatment with PNGase F, an amidase widely used to remove N-linked glycans from glycoproteins, suggested that BC15/OsCTL1 is N-glycosylated (Fig. 3D).
		+
		+	[[File:three.jpg]]

	===Evolution===		===Evolution===

← Older revision		Revision as of 13:34, 3 June 2014
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	[[File:two.jpg]]		[[File:two.jpg]]
−

	===Structure===		===Structure===

← Older revision		Revision as of 13:33, 3 June 2014
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	[[File:two.jpg]]		[[File:two.jpg]]
		+
		+
		+	===Structure===

	===Evolution===		===Evolution===

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 <references>
 <ref name="ref1">Wu B, et al. Brittle culm15 encodes a membrane-associated chitinase-like protein required for cellulose biosynthesis in rice[J]. Plant Physiol, 2012, 159(4):1440-52.</ref>
-<ref name="ref2">Rao Y C, et al. Characterization and cloning of a brittle culm  mutant (bc88 ) in rice ( Oryza sativa L.)[J]. Chin Sci Bull, 2013, 58:
+<ref name="ref2">Rao Y C, et al. Characterization and cloning of a brittle culm  mutant (bc88) in rice ( Oryza sativa L.)[J]. Chin Sci Bull, 2013, 58:
 -3006.</ref>
 </references>