Report Clarification

Kanzaki, Makoto; Nagasawa, Mitsuru; Kojima, Itaru; Sato, Chikara; Naruse, Keiji; Sokabe, Masahiro; Iida, Hidetoshi

doi:10.1126/science.288.5470.1347

Cited by 18 publications

(18 citation statements)

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“…In addition, our recent study with protease protection experiments has shown that GFP in the Mid1-GFP fusion protein, in which GFP has been fused downstream from H3, is susceptible to protease added to intact yeast cells. 3 In contrast, GFP in the Mid1-GFP fusion protein, in which GFP has been fused downstream from H4, is resistant to protease as expected. 3 These results suggest that H4 is a transmembrane segment and that Phe 356 is outside the plasma membrane.…”

Section: Important Amino Acid Residues In H3mentioning

confidence: 99%

“…3 In contrast, GFP in the Mid1-GFP fusion protein, in which GFP has been fused downstream from H4, is resistant to protease as expected. 3 These results suggest that H4 is a transmembrane segment and that Phe 356 is outside the plasma membrane. Therefore, we speculate that Phe 356 interacts with an extracellular portion of other protein(s) which takes part in the maintenance of cell viability without regulating Ca 2ϩ uptake.…”

Section: Important Amino Acid Residues In H3mentioning

confidence: 99%

“…It has also been shown that the Mid1 protein localizes at the endoplasmic reticulum (ER) membrane as well as the plasma membrane, as revealed by indirect fluorescence microscopy. 2 The wild-type Mid1-GFP produced from low-and multicopy plasmids has been shown to completely complement the mid phenotype and to localize at the plasma membrane and endoplasmic reticulum membrane normally, 3 indicating that GFP and the overexpression has no effect on the activity and localization of the Mid1 protein. However, it should be noted that fluorescence images of Mid1-GFP produced from the low copy plasmid were very faint and hard to be photographed.…”

Section: H3 and H4 May Not Be Required For The Localization Of The MImentioning

confidence: 99%

“…However, it should be noted that fluorescence images of Mid1-GFP produced from the low copy plasmid were very faint and hard to be photographed. 3 In addition, we have tried to examine the localization of the Mid1 protein produced from its gene on the intrinsic chromosome or on a low copy plasmid by indirect fluorescence microscopy using polyclonal anti-Mid1 antibodies, but we failed because of the very small expression levels of Mid1. We therefore examined the localization of Mid1-GFP produced from the multicopy plasmid thereafter.…”

Section: H3 and H4 May Not Be Required For The Localization Of The MImentioning

confidence: 99%

“…This phenotype is called the mid phenotype for the mating pheromone-induced death phenotype. Electrophysiological and cell biological studies on Chinese hamster ovary cells, Balb/c 3T3 cells, and COS-7 cells expressing Mid1 have revealed that it functions as a Ca 2ϩ -permeable stretch-activated channel (2,3). The mating pheromone leads their target cells to differentiate into cells (so-called "shmoos") having a mating projection at which the plasma membrane and the cell wall are degraded and regenerated (4,5).…”

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confidence: 99%

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Molecular Dissection of the Hydrophobic Segments H3 and H4 of the Yeast Ca2+ Channel Component Mid1

Tada¹,

Ohmori²,

Iida³

2003

Journal of Biological Chemistry

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The Saccharomyces cerevisiae MID1 gene product, Mid1, is composed of 548 amino acid residues, has four relatively hydrophobic segments named H1-H4, and functions as a Ca 2؉ -permeable, stretch-activated channel when expressed in mammalian cells. In some conditions Mid1 cooperates with Cch1, a yeast homolog of the ␣1 subunit of mammalian voltage-gated channels. To identify the important regions or amino acid residues necessary for Mid1 function, we employed in vitro sitedirected mutagenesis on H3 and H4 of Mid1 and expressed the resulting mutant genes in a mid1 null mutant to examine whether the mutant gene products are functional or not in vivo. Mutant Mid1 proteins lacking the whole H3 or H4 segment, H3De or H4De, did not complement the lethality and low Ca 2؉ accumulation activity of the mid1 mutant, although their localization and contents appeared to be normal, indicating that H3 and H4 are required for Mid1 function itself. Single amino acid exchange experiments on individual amino acid residues of H3 and H4 showed that 10 of 20 residues in H3 and 14 of 23 residues in H4 were important for the normal function of Mid1. In particular, we found four severe loss-of-function mutations, D341E, F356S, C373D, and C373R, and two interesting mutations leading to a high level of Ca 2؉ accumulation with a slightly low complementing activity, G342A and Y355A. The importance of these amino acid residues will be discussed.

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Section: Important Amino Acid Residues In H3mentioning

confidence: 99%

Section: Important Amino Acid Residues In H3mentioning

confidence: 99%

Section: H3 and H4 May Not Be Required For The Localization Of The MImentioning

confidence: 99%

Section: H3 and H4 May Not Be Required For The Localization Of The MImentioning

confidence: 99%

mentioning

confidence: 99%

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Molecular Dissection of the Hydrophobic Segments H3 and H4 of the Yeast Ca2+ Channel Component Mid1

Tada¹,

Ohmori²,

Iida³

2003

Journal of Biological Chemistry

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Involvement of the putative Ca2+-permeable mechanosensitive channels, NtMCA1 and NtMCA2, in Ca2+ uptake, Ca2+-dependent cell proliferation and mechanical stress-induced gene expression in tobacco (Nicotiana tabacum) BY-2 cells

et al. 2011

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To gain insight into the cellular functions of the mid1-complementing activity (MCA) family proteins, encoding putative Ca²⁺-permeable mechanosensitive channels, we isolated two MCA homologs of tobacco (Nicotiana tabacum) BY-2 cells, named NtMCA1 and NtMCA2. NtMCA1 and NtMCA2 partially complemented the lethality and Ca²⁺ uptake defects of yeast mutants lacking mechanosensitive Ca²⁺ channel components. Furthermore, in yeast cells overexpressing NtMCA1 and NtMCA2, the hypo-osmotic shock-induced Ca²⁺ influx was enhanced. Overexpression of NtMCA1 or NtMCA2 in BY-2 cells enhanced Ca²⁺ uptake, and significantly alleviated growth inhibition under Ca²⁺ limitation. NtMCA1-overexpressing BY-2 cells showed higher sensitivity to hypo-osmotic shock than control cells, and induced the expression of the touch-inducible gene, NtERF4. We found that both NtMCA1-GFP and NtMCA2-GFP were localized at the plasma membrane and its interface with the cell wall, Hechtian strands, and at the cell plate and perinuclear vesicles of dividing cells. NtMCA2 transcript levels fluctuated during the cell cycle and were highest at the G1 phase. These results suggest that NtMCA1 and NtMCA2 play roles in Ca²⁺-dependent cell proliferation and mechanical stress-induced gene expression in BY-2 cells, by regulating the Ca²⁺ influx through the plasma membrane.

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6.5 Mechanosensory Transduction

Martinac

Kloda²

2012

Comprehensive Biophysics

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Report Clarification

Cited by 18 publications

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Molecular Dissection of the Hydrophobic Segments H3 and H4 of the Yeast Ca2+ Channel Component Mid1

Molecular Dissection of the Hydrophobic Segments H3 and H4 of the Yeast Ca2+ Channel Component Mid1

Involvement of the putative Ca2+-permeable mechanosensitive channels, NtMCA1 and NtMCA2, in Ca2+ uptake, Ca2+-dependent cell proliferation and mechanical stress-induced gene expression in tobacco (Nicotiana tabacum) BY-2 cells

6.5 Mechanosensory Transduction

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