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The intermediate filament protein peripherin is the specific interaction partner of mouse BPAG1-n (dystonin) in neurons.

Leung CL, Sun D, Liem RK - J. Cell Biol. (1999)

Bottom Line: Kouklis, D.W.Fuchs. 1996.Since peripherin and BPAG1-n also display similar expression patterns in the nervous system, we suggest that peripherin is the specific interaction partner of BPAG1-n in vivo.

View Article: PubMed Central - PubMed

Affiliation: Departments of Biochemistry and Molecular Biophysics, Columbia University College of Physicians and Surgeons, New York 10032, USA.

ABSTRACT
The dystonia musculorum (dt) mouse suffers from severe degeneration of primary sensory neurons. The mutated gene product is named dystonin and is identical to the neuronal isoform of bullous pemphigoid antigen 1 (BPAG1-n). BPAG1-n contains an actin-binding domain at its NH2 terminus and a putative intermediate filament-binding domain at its COOH terminus. Because the degenerating sensory neurons of dt mice display abnormal accumulations of intermediate filaments in the axons, BPAG1-n has been postulated to organize the neuronal cytoskeleton by interacting with both the neurofilament triplet proteins (NFTPs) and microfilaments. In this paper we show by a variety of methods that the COOH-terminal tail domain of mouse BPAG1 interacts specifically with peripherin, but in contrast to a previous study (Yang, Y., J. Dowling, Q.C. Yu, P. Kouklis, D.W. Cleveland, and E. Fuchs. 1996. Cell. 86:655-665), mouse BPAG1 fails to associate with full-length NFTPs. The tail domains interfered with the association of the NFTPs with BPAG1. In dt mice, peripherin is present in axonal swellings of degenerating sensory neurons in the dorsal root ganglia and is downregulated even in other neural regions, which have no obvious signs of pathology. Since peripherin and BPAG1-n also display similar expression patterns in the nervous system, we suggest that peripherin is the specific interaction partner of BPAG1-n in vivo.

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Three-hybrid analysis of interactions between  mBPAG-C1 and NFTPs. Three-hybrid vectors containing two  expression cassettes of GAL4 transactivation domain fused to  NFTPs were cotransformed with pGBT-mBPAG-C1 in SFY526  yeast cells. Cells were grown to mid-log phase in minus Trp-Leu  media and permeabilized for quantitative β-galactosidase assays  as described in the text. LΔ, MΔ, and HΔ refer to the tailless proteins NFL1-415, NFM1-421, and NFH1-415, respectively.
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Figure 6: Three-hybrid analysis of interactions between mBPAG-C1 and NFTPs. Three-hybrid vectors containing two expression cassettes of GAL4 transactivation domain fused to NFTPs were cotransformed with pGBT-mBPAG-C1 in SFY526 yeast cells. Cells were grown to mid-log phase in minus Trp-Leu media and permeabilized for quantitative β-galactosidase assays as described in the text. LΔ, MΔ, and HΔ refer to the tailless proteins NFL1-415, NFM1-421, and NFH1-415, respectively.

Mentions: Since we have shown that NF-H and NF-M prefer to form heterodimers with NF-L (Leung and Liem, 1996), we performed three-hybrid studies to examine whether mBPAG-C1 could associate with heterodimers of NF-L/NF-M (L/M) or NF-L/NF-H (L/H). We took advantage of the yeast strain HF7c, which contained a HIS3 reporter gene, as well as a weak lacZ reporter gene. We reasoned that if mBPAG-C1 could interact with heterodimers of L/M or L/H, triple transformants of pGBT-mBPAG-C1/pGAD-NFL/pGAD-NFM or pGBT-mBPAG-C1/pGAD-NFL/ pGAD-NFH in HF7c would grow on minus histidine-leucine-tryptophan synthetic dropout plates. In addition, the selective pressure of histidine-free medium would keep all three plasmids in the same cells, provided that the expressed proteins interacted together as a unit. Under these conditions, there were still no interactions between mBPAG-C1 and L/M or L/H (Table II). Therefore, we postulated that the failure of NFTPs to interact with mBPAG-C1 might be inherent in the structure of the molecules. Since the greatest differences among nIF proteins are at their tail domains, we prepared tailless NFTPs (LΔ, MΔ, and HΔ) and tested for their interactions with mBPAG-C1 in yeast strain HF7c. We observed fast growing colonies in triple transformants of pGBT-mBPAG-C1/ pGAD-LΔ/pGAD-HΔ. Some of these colonies also displayed weak β-galactosidase activities. These results imply that mBPAG-C1 interacted with heterodimers of tailless NF-L and NF-H (Table II). However, this interpretation was tempered, because very slow growing small colonies were also detected in cotransformants of pGBT-mBPAG-C1 with any one of the tailless GAD-NFTP fusion vectors, indicating weak interactions between the tailless NFTPs and mBPAG-C1 (Table II). To evaluate whether mBPAG- C1 really preferred to bind to the heterodimer of tailless NF-L and tailless NF-H, we constructed pGAD424-based three-hybrid vectors that contained two expression cassettes. By these means, two GAD fusion proteins could be expressed in the same transformant with single selection. Cotransformants of the three-hybrid vectors and pGBT-mBPAG-C1 would express the three fusion proteins simultaneously. Quantitative measurements of β-galactosidase activities were used to determine the relative strength of each interaction. The yeast transformants with pGBT-mBPAG-C1/pGAD-LΔ/pGAD-HΔ consistently displayed a high β-galactosidase activity, further confirming that mBPAG-C1 interact strongly with the LΔ/HΔ heterodimer (Fig. 6).


The intermediate filament protein peripherin is the specific interaction partner of mouse BPAG1-n (dystonin) in neurons.

Leung CL, Sun D, Liem RK - J. Cell Biol. (1999)

Three-hybrid analysis of interactions between  mBPAG-C1 and NFTPs. Three-hybrid vectors containing two  expression cassettes of GAL4 transactivation domain fused to  NFTPs were cotransformed with pGBT-mBPAG-C1 in SFY526  yeast cells. Cells were grown to mid-log phase in minus Trp-Leu  media and permeabilized for quantitative β-galactosidase assays  as described in the text. LΔ, MΔ, and HΔ refer to the tailless proteins NFL1-415, NFM1-421, and NFH1-415, respectively.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2132913&req=5

Figure 6: Three-hybrid analysis of interactions between mBPAG-C1 and NFTPs. Three-hybrid vectors containing two expression cassettes of GAL4 transactivation domain fused to NFTPs were cotransformed with pGBT-mBPAG-C1 in SFY526 yeast cells. Cells were grown to mid-log phase in minus Trp-Leu media and permeabilized for quantitative β-galactosidase assays as described in the text. LΔ, MΔ, and HΔ refer to the tailless proteins NFL1-415, NFM1-421, and NFH1-415, respectively.
Mentions: Since we have shown that NF-H and NF-M prefer to form heterodimers with NF-L (Leung and Liem, 1996), we performed three-hybrid studies to examine whether mBPAG-C1 could associate with heterodimers of NF-L/NF-M (L/M) or NF-L/NF-H (L/H). We took advantage of the yeast strain HF7c, which contained a HIS3 reporter gene, as well as a weak lacZ reporter gene. We reasoned that if mBPAG-C1 could interact with heterodimers of L/M or L/H, triple transformants of pGBT-mBPAG-C1/pGAD-NFL/pGAD-NFM or pGBT-mBPAG-C1/pGAD-NFL/ pGAD-NFH in HF7c would grow on minus histidine-leucine-tryptophan synthetic dropout plates. In addition, the selective pressure of histidine-free medium would keep all three plasmids in the same cells, provided that the expressed proteins interacted together as a unit. Under these conditions, there were still no interactions between mBPAG-C1 and L/M or L/H (Table II). Therefore, we postulated that the failure of NFTPs to interact with mBPAG-C1 might be inherent in the structure of the molecules. Since the greatest differences among nIF proteins are at their tail domains, we prepared tailless NFTPs (LΔ, MΔ, and HΔ) and tested for their interactions with mBPAG-C1 in yeast strain HF7c. We observed fast growing colonies in triple transformants of pGBT-mBPAG-C1/ pGAD-LΔ/pGAD-HΔ. Some of these colonies also displayed weak β-galactosidase activities. These results imply that mBPAG-C1 interacted with heterodimers of tailless NF-L and NF-H (Table II). However, this interpretation was tempered, because very slow growing small colonies were also detected in cotransformants of pGBT-mBPAG-C1 with any one of the tailless GAD-NFTP fusion vectors, indicating weak interactions between the tailless NFTPs and mBPAG-C1 (Table II). To evaluate whether mBPAG- C1 really preferred to bind to the heterodimer of tailless NF-L and tailless NF-H, we constructed pGAD424-based three-hybrid vectors that contained two expression cassettes. By these means, two GAD fusion proteins could be expressed in the same transformant with single selection. Cotransformants of the three-hybrid vectors and pGBT-mBPAG-C1 would express the three fusion proteins simultaneously. Quantitative measurements of β-galactosidase activities were used to determine the relative strength of each interaction. The yeast transformants with pGBT-mBPAG-C1/pGAD-LΔ/pGAD-HΔ consistently displayed a high β-galactosidase activity, further confirming that mBPAG-C1 interact strongly with the LΔ/HΔ heterodimer (Fig. 6).

Bottom Line: Kouklis, D.W.Fuchs. 1996.Since peripherin and BPAG1-n also display similar expression patterns in the nervous system, we suggest that peripherin is the specific interaction partner of BPAG1-n in vivo.

View Article: PubMed Central - PubMed

Affiliation: Departments of Biochemistry and Molecular Biophysics, Columbia University College of Physicians and Surgeons, New York 10032, USA.

ABSTRACT
The dystonia musculorum (dt) mouse suffers from severe degeneration of primary sensory neurons. The mutated gene product is named dystonin and is identical to the neuronal isoform of bullous pemphigoid antigen 1 (BPAG1-n). BPAG1-n contains an actin-binding domain at its NH2 terminus and a putative intermediate filament-binding domain at its COOH terminus. Because the degenerating sensory neurons of dt mice display abnormal accumulations of intermediate filaments in the axons, BPAG1-n has been postulated to organize the neuronal cytoskeleton by interacting with both the neurofilament triplet proteins (NFTPs) and microfilaments. In this paper we show by a variety of methods that the COOH-terminal tail domain of mouse BPAG1 interacts specifically with peripherin, but in contrast to a previous study (Yang, Y., J. Dowling, Q.C. Yu, P. Kouklis, D.W. Cleveland, and E. Fuchs. 1996. Cell. 86:655-665), mouse BPAG1 fails to associate with full-length NFTPs. The tail domains interfered with the association of the NFTPs with BPAG1. In dt mice, peripherin is present in axonal swellings of degenerating sensory neurons in the dorsal root ganglia and is downregulated even in other neural regions, which have no obvious signs of pathology. Since peripherin and BPAG1-n also display similar expression patterns in the nervous system, we suggest that peripherin is the specific interaction partner of BPAG1-n in vivo.

Show MeSH
Related in: MedlinePlus