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Tts1, the fission yeast homologue of the TMEM33 family, functions in NE remodeling during mitosis.

Zhang D, Oliferenko S - Mol. Biol. Cell (2014)

Bottom Line: An amphipathic helix located at the C-terminus of Tts1 is important for ER shaping and modulating the mitotic NPC distribution.Of interest, the evolutionarily conserved residues at the luminal interface of the third transmembrane region function specifically in promoting SPB-NE insertion.Our data illuminate cellular requirements for remodeling the NE during "closed" nuclear division and provide insight into the structure and functions of the eukaryotic TMEM33 family.

View Article: PubMed Central - PubMed

Affiliation: Temasek Life Sciences Laboratory, National University of Singapore, Singapore 117604 zhangdan@tll.org.sg snezhana.oliferenko@kcl.ac.uk.

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Evolutionarily conserved motifs in Tts1 are required for its partitioning to the tubular ER. (A) Predicted structural features and conserved motifs in the TMEM33 protein Tts1 in S. pombe. Note that the motif shown in red is conserved throughout the TMEM33 family, whereas the motif in blue contains two charged arginines that are highly conserved among its fungal orthologues. Highlighted are conserved residues that were mutated in this study. The helical wheel showing that helix 3 in Tts1 C-terminus potentially has amphipathic properties was generated using HeliQuest (http://heliquest.ipmc.cnrs.fr/). Mutated residues at the hydrophobic side are indicated by red points. (B) Localization of GFP-fused Tts1 mutants in tts1Δ cells expressing Rtn1-mCherry. The cartoons show the logic of construction of Tts1 mutants. Scanning confocal micrographs of cells expressing indicated proteins were taken from either top or middle (for Tts1-Cter) planes of z-stacks. Scale bars, 5 μm. Normalized colocalization factors shown in red indicate the significant decrease of specificity in the tubular ER localization of the corresponding Tts1 mutants (mean ± SD; 10 < n < 35). *p ≪ 10−4 in comparison to the wild type; two-tailed Student's t test.
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Figure 1: Evolutionarily conserved motifs in Tts1 are required for its partitioning to the tubular ER. (A) Predicted structural features and conserved motifs in the TMEM33 protein Tts1 in S. pombe. Note that the motif shown in red is conserved throughout the TMEM33 family, whereas the motif in blue contains two charged arginines that are highly conserved among its fungal orthologues. Highlighted are conserved residues that were mutated in this study. The helical wheel showing that helix 3 in Tts1 C-terminus potentially has amphipathic properties was generated using HeliQuest (http://heliquest.ipmc.cnrs.fr/). Mutated residues at the hydrophobic side are indicated by red points. (B) Localization of GFP-fused Tts1 mutants in tts1Δ cells expressing Rtn1-mCherry. The cartoons show the logic of construction of Tts1 mutants. Scanning confocal micrographs of cells expressing indicated proteins were taken from either top or middle (for Tts1-Cter) planes of z-stacks. Scale bars, 5 μm. Normalized colocalization factors shown in red indicate the significant decrease of specificity in the tubular ER localization of the corresponding Tts1 mutants (mean ± SD; 10 < n < 35). *p ≪ 10−4 in comparison to the wild type; two-tailed Student's t test.

Mentions: Sequence analyses suggest that Tts1 has four transmembrane (TM) regions in the N-terminal half of the protein, followed by a string of three α-helices exposed to the cytosol (Figure 1A; predictions of topology are consistent between www.cbs.dtu.dk/services/TMHMM/ and www.enzim.hu/hmmtop/index.php; Tusnady and Simon, 2001; C-terminal helix predictions are based on Petersen et al., 2009; www.cbs.dtu.dk/services/NetSurfP/). Sequence alignment among proteins from the eukaryotic TMEM33 family revealed two highly conserved motifs (Figure 1A; Bailey et al., 2009; http://meme.nbcr.net/meme/). One motif was found at the luminal interface of the third TM stretch, with a conserved Pro-119 and three aromatic side-chain residues (Tyr-123, His-127, and Tyr-131) predicted to lie on the same side of the helix (Figure 1A; highlighted in yellow). The other conserved, nine–amino acid motif, delimited by two tyrosines (Tyr-203 and Tyr-211), resides in the first of the three cytosolic α-helices (Figure 1A). The positively charged residues (Arg-208/210) within this motif are conserved in fungi (Figure 1A). Although not conserved outside the fission yeast clade, the third C-terminal helix from Val-240 to Ala-257 (VIKNAWHTFKTYVSKFGA) is predicted to exhibit amphipathic properties (Figure 1A; Gautier et al., 2008; http://heliquest.ipmc.cnrs.fr/).


Tts1, the fission yeast homologue of the TMEM33 family, functions in NE remodeling during mitosis.

Zhang D, Oliferenko S - Mol. Biol. Cell (2014)

Evolutionarily conserved motifs in Tts1 are required for its partitioning to the tubular ER. (A) Predicted structural features and conserved motifs in the TMEM33 protein Tts1 in S. pombe. Note that the motif shown in red is conserved throughout the TMEM33 family, whereas the motif in blue contains two charged arginines that are highly conserved among its fungal orthologues. Highlighted are conserved residues that were mutated in this study. The helical wheel showing that helix 3 in Tts1 C-terminus potentially has amphipathic properties was generated using HeliQuest (http://heliquest.ipmc.cnrs.fr/). Mutated residues at the hydrophobic side are indicated by red points. (B) Localization of GFP-fused Tts1 mutants in tts1Δ cells expressing Rtn1-mCherry. The cartoons show the logic of construction of Tts1 mutants. Scanning confocal micrographs of cells expressing indicated proteins were taken from either top or middle (for Tts1-Cter) planes of z-stacks. Scale bars, 5 μm. Normalized colocalization factors shown in red indicate the significant decrease of specificity in the tubular ER localization of the corresponding Tts1 mutants (mean ± SD; 10 < n < 35). *p ≪ 10−4 in comparison to the wild type; two-tailed Student's t test.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Figure 1: Evolutionarily conserved motifs in Tts1 are required for its partitioning to the tubular ER. (A) Predicted structural features and conserved motifs in the TMEM33 protein Tts1 in S. pombe. Note that the motif shown in red is conserved throughout the TMEM33 family, whereas the motif in blue contains two charged arginines that are highly conserved among its fungal orthologues. Highlighted are conserved residues that were mutated in this study. The helical wheel showing that helix 3 in Tts1 C-terminus potentially has amphipathic properties was generated using HeliQuest (http://heliquest.ipmc.cnrs.fr/). Mutated residues at the hydrophobic side are indicated by red points. (B) Localization of GFP-fused Tts1 mutants in tts1Δ cells expressing Rtn1-mCherry. The cartoons show the logic of construction of Tts1 mutants. Scanning confocal micrographs of cells expressing indicated proteins were taken from either top or middle (for Tts1-Cter) planes of z-stacks. Scale bars, 5 μm. Normalized colocalization factors shown in red indicate the significant decrease of specificity in the tubular ER localization of the corresponding Tts1 mutants (mean ± SD; 10 < n < 35). *p ≪ 10−4 in comparison to the wild type; two-tailed Student's t test.
Mentions: Sequence analyses suggest that Tts1 has four transmembrane (TM) regions in the N-terminal half of the protein, followed by a string of three α-helices exposed to the cytosol (Figure 1A; predictions of topology are consistent between www.cbs.dtu.dk/services/TMHMM/ and www.enzim.hu/hmmtop/index.php; Tusnady and Simon, 2001; C-terminal helix predictions are based on Petersen et al., 2009; www.cbs.dtu.dk/services/NetSurfP/). Sequence alignment among proteins from the eukaryotic TMEM33 family revealed two highly conserved motifs (Figure 1A; Bailey et al., 2009; http://meme.nbcr.net/meme/). One motif was found at the luminal interface of the third TM stretch, with a conserved Pro-119 and three aromatic side-chain residues (Tyr-123, His-127, and Tyr-131) predicted to lie on the same side of the helix (Figure 1A; highlighted in yellow). The other conserved, nine–amino acid motif, delimited by two tyrosines (Tyr-203 and Tyr-211), resides in the first of the three cytosolic α-helices (Figure 1A). The positively charged residues (Arg-208/210) within this motif are conserved in fungi (Figure 1A). Although not conserved outside the fission yeast clade, the third C-terminal helix from Val-240 to Ala-257 (VIKNAWHTFKTYVSKFGA) is predicted to exhibit amphipathic properties (Figure 1A; Gautier et al., 2008; http://heliquest.ipmc.cnrs.fr/).

Bottom Line: An amphipathic helix located at the C-terminus of Tts1 is important for ER shaping and modulating the mitotic NPC distribution.Of interest, the evolutionarily conserved residues at the luminal interface of the third transmembrane region function specifically in promoting SPB-NE insertion.Our data illuminate cellular requirements for remodeling the NE during "closed" nuclear division and provide insight into the structure and functions of the eukaryotic TMEM33 family.

View Article: PubMed Central - PubMed

Affiliation: Temasek Life Sciences Laboratory, National University of Singapore, Singapore 117604 zhangdan@tll.org.sg snezhana.oliferenko@kcl.ac.uk.

Show MeSH
Related in: MedlinePlus