Limits...
Global defects in collagen secretion in a Mia3/TANGO1 knockout mouse.

Wilson DG, Phamluong K, Li L, Sun M, Cao TC, Liu PS, Modrusan Z, Sandoval WN, Rangell L, Carano RA, Peterson AS, Solloway MJ - J. Cell Biol. (2011)

Bottom Line: These changes are associated with intracellular accumulation of collagen and the induction of a strong unfolded protein response, primarily within the developing skeleton.Chondrocyte maturation and bone mineralization are severely compromised in Mia3- embryos, leading to dwarfism and neonatal lethality.Thus, Mia3's role in protein secretion is much broader than previously realized, and it may, in fact, be required for the efficient secretion of all collagen molecules in higher organisms.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Molecular Biology, Genentech, South San Francisco, CA 94080, USA.

ABSTRACT
Melanoma inhibitory activity member 3 (MIA3/TANGO1) [corrected] is an evolutionarily conserved endoplasmic reticulum resident transmembrane protein. Recent in vitro studies have shown that it is required for the loading of collagen VII, but not collagen I, into COPII-coated transport vesicles. In this paper, we show that mice lacking Mia3 are defective for the secretion of numerous collagens, including collagens I, II, III, IV, VII, and IX, from chondrocytes, fibroblasts, endothelial cells, and mural cells. Collagen deposition by these cell types is abnormal, and extracellular matrix composition is compromised. These changes are associated with intracellular accumulation of collagen and the induction of a strong unfolded protein response, primarily within the developing skeleton. Chondrocyte maturation and bone mineralization are severely compromised in Mia3- embryos, leading to dwarfism and neonatal lethality. Thus, Mia3's role in protein secretion is much broader than previously realized, and it may, in fact, be required for the efficient secretion of all collagen molecules in higher organisms.

Show MeSH

Related in: MedlinePlus

Mia3 is an ER-associated protein. (A) Hydropathy chart, exon alignment, and proposed domain structure of Mia3 with two antibody (α-Mia3) epitopes indicated. SP, signal peptide; SH3, putative SH3-like fold; TM, transmembrane domain; PRD, proline-rich domain; pred. MM, predicted molecular mass. (B and C) Mia3 intron/exon map and description of the targeted allele. Nucleotides highlighted in red correspond to exon2 and exon3 sequences. LacZ/neo, LacZ/neomycin. (D) α-Mia3 SH3 polyclonal antibodies show punctate intracellular staining that is absent in Mia3- MEFs or nonpermeabilized cells. (E) Western blot analysis of total protein, membrane (membr), and cytosolic (cyto) fractions from wt and Mia3−/− 14.5-dpc embryos reveals two major bands near 250 kD with several smaller isoforms or degradation products in the membrane fraction. NS, nonspecific antibody cross-reactivity; C-term, C terminus; KO, knockout. Molecular masses are given in kilodaltons. (F) Immunofluorescent colocalization analyses of α-Mia3 SH3 with antibodies against calnexin, Hsp47 (SerpinH1), ERGIC-53 (Lman1), and GM130 in primary chondrocytes reveals an Mia3 protein within punctate structures on the ER membrane. Insets detail regions boxed in red. Bars, 10 µm.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC3105544&req=5

fig1: Mia3 is an ER-associated protein. (A) Hydropathy chart, exon alignment, and proposed domain structure of Mia3 with two antibody (α-Mia3) epitopes indicated. SP, signal peptide; SH3, putative SH3-like fold; TM, transmembrane domain; PRD, proline-rich domain; pred. MM, predicted molecular mass. (B and C) Mia3 intron/exon map and description of the targeted allele. Nucleotides highlighted in red correspond to exon2 and exon3 sequences. LacZ/neo, LacZ/neomycin. (D) α-Mia3 SH3 polyclonal antibodies show punctate intracellular staining that is absent in Mia3- MEFs or nonpermeabilized cells. (E) Western blot analysis of total protein, membrane (membr), and cytosolic (cyto) fractions from wt and Mia3−/− 14.5-dpc embryos reveals two major bands near 250 kD with several smaller isoforms or degradation products in the membrane fraction. NS, nonspecific antibody cross-reactivity; C-term, C terminus; KO, knockout. Molecular masses are given in kilodaltons. (F) Immunofluorescent colocalization analyses of α-Mia3 SH3 with antibodies against calnexin, Hsp47 (SerpinH1), ERGIC-53 (Lman1), and GM130 in primary chondrocytes reveals an Mia3 protein within punctate structures on the ER membrane. Insets detail regions boxed in red. Bars, 10 µm.

Mentions: To elucidate the role of MIA3 in vivo, we characterized the phenotype of a Mia3 knockout mouse. Mia3 contains a putative signal peptide, an N-terminal SH3 domain followed by two coiled-coil domains, a transmembrane domain, and a C-terminal proline-rich domain (Fig. 1 A). A gene-targeting cassette encoding a LacZ/neomycin fusion protein was inserted in frame 11 bases into the beginning of the second exon, replacing the contents of this exon and all of exon3 (Fig. 1, B and C). This vector deletes the SH3 domain that has been shown to mediate interactions with Col7a1 (Saito et al., 2009b). Correct targeting events were confirmed by Southern blot analysis using both 5′ and 3′ genomic probes as well as PCR (Fig. S1 A).


Global defects in collagen secretion in a Mia3/TANGO1 knockout mouse.

Wilson DG, Phamluong K, Li L, Sun M, Cao TC, Liu PS, Modrusan Z, Sandoval WN, Rangell L, Carano RA, Peterson AS, Solloway MJ - J. Cell Biol. (2011)

Mia3 is an ER-associated protein. (A) Hydropathy chart, exon alignment, and proposed domain structure of Mia3 with two antibody (α-Mia3) epitopes indicated. SP, signal peptide; SH3, putative SH3-like fold; TM, transmembrane domain; PRD, proline-rich domain; pred. MM, predicted molecular mass. (B and C) Mia3 intron/exon map and description of the targeted allele. Nucleotides highlighted in red correspond to exon2 and exon3 sequences. LacZ/neo, LacZ/neomycin. (D) α-Mia3 SH3 polyclonal antibodies show punctate intracellular staining that is absent in Mia3- MEFs or nonpermeabilized cells. (E) Western blot analysis of total protein, membrane (membr), and cytosolic (cyto) fractions from wt and Mia3−/− 14.5-dpc embryos reveals two major bands near 250 kD with several smaller isoforms or degradation products in the membrane fraction. NS, nonspecific antibody cross-reactivity; C-term, C terminus; KO, knockout. Molecular masses are given in kilodaltons. (F) Immunofluorescent colocalization analyses of α-Mia3 SH3 with antibodies against calnexin, Hsp47 (SerpinH1), ERGIC-53 (Lman1), and GM130 in primary chondrocytes reveals an Mia3 protein within punctate structures on the ER membrane. Insets detail regions boxed in red. Bars, 10 µm.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3105544&req=5

fig1: Mia3 is an ER-associated protein. (A) Hydropathy chart, exon alignment, and proposed domain structure of Mia3 with two antibody (α-Mia3) epitopes indicated. SP, signal peptide; SH3, putative SH3-like fold; TM, transmembrane domain; PRD, proline-rich domain; pred. MM, predicted molecular mass. (B and C) Mia3 intron/exon map and description of the targeted allele. Nucleotides highlighted in red correspond to exon2 and exon3 sequences. LacZ/neo, LacZ/neomycin. (D) α-Mia3 SH3 polyclonal antibodies show punctate intracellular staining that is absent in Mia3- MEFs or nonpermeabilized cells. (E) Western blot analysis of total protein, membrane (membr), and cytosolic (cyto) fractions from wt and Mia3−/− 14.5-dpc embryos reveals two major bands near 250 kD with several smaller isoforms or degradation products in the membrane fraction. NS, nonspecific antibody cross-reactivity; C-term, C terminus; KO, knockout. Molecular masses are given in kilodaltons. (F) Immunofluorescent colocalization analyses of α-Mia3 SH3 with antibodies against calnexin, Hsp47 (SerpinH1), ERGIC-53 (Lman1), and GM130 in primary chondrocytes reveals an Mia3 protein within punctate structures on the ER membrane. Insets detail regions boxed in red. Bars, 10 µm.
Mentions: To elucidate the role of MIA3 in vivo, we characterized the phenotype of a Mia3 knockout mouse. Mia3 contains a putative signal peptide, an N-terminal SH3 domain followed by two coiled-coil domains, a transmembrane domain, and a C-terminal proline-rich domain (Fig. 1 A). A gene-targeting cassette encoding a LacZ/neomycin fusion protein was inserted in frame 11 bases into the beginning of the second exon, replacing the contents of this exon and all of exon3 (Fig. 1, B and C). This vector deletes the SH3 domain that has been shown to mediate interactions with Col7a1 (Saito et al., 2009b). Correct targeting events were confirmed by Southern blot analysis using both 5′ and 3′ genomic probes as well as PCR (Fig. S1 A).

Bottom Line: These changes are associated with intracellular accumulation of collagen and the induction of a strong unfolded protein response, primarily within the developing skeleton.Chondrocyte maturation and bone mineralization are severely compromised in Mia3- embryos, leading to dwarfism and neonatal lethality.Thus, Mia3's role in protein secretion is much broader than previously realized, and it may, in fact, be required for the efficient secretion of all collagen molecules in higher organisms.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Molecular Biology, Genentech, South San Francisco, CA 94080, USA.

ABSTRACT
Melanoma inhibitory activity member 3 (MIA3/TANGO1) [corrected] is an evolutionarily conserved endoplasmic reticulum resident transmembrane protein. Recent in vitro studies have shown that it is required for the loading of collagen VII, but not collagen I, into COPII-coated transport vesicles. In this paper, we show that mice lacking Mia3 are defective for the secretion of numerous collagens, including collagens I, II, III, IV, VII, and IX, from chondrocytes, fibroblasts, endothelial cells, and mural cells. Collagen deposition by these cell types is abnormal, and extracellular matrix composition is compromised. These changes are associated with intracellular accumulation of collagen and the induction of a strong unfolded protein response, primarily within the developing skeleton. Chondrocyte maturation and bone mineralization are severely compromised in Mia3- embryos, leading to dwarfism and neonatal lethality. Thus, Mia3's role in protein secretion is much broader than previously realized, and it may, in fact, be required for the efficient secretion of all collagen molecules in higher organisms.

Show MeSH
Related in: MedlinePlus