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K Matsuoka - One of the best experts on this subject based on the ideXlab platform.

  • surface structure of the copii coated Vesicle
    Proceedings of the National Academy of Sciences of the United States of America, 2001
    Co-Authors: K Matsuoka, Lelio Orci, John E. Heuser
    Abstract:

    The spatial arrangement of COPII coat protein subunits was analyzed by crosslinking to an artificial membrane surface and by electron microscopy of coat proteins and coated Vesicle surfaces. The efficiency of COPII subunit crosslinking to phospholipids declined in order of protein recruitment to the coat: Sar1p > Sec23/24p ≫ Sec13/31p. Deep-etch rotary shadowing and electron microscopy were used to explore the COPII subunit structure with isolated proteins and coated Vesicles. Sec23/24 resembles a bow tie, and Sec13/31p contains terminal bilobed globular structures bordering a central rod. The surface structure of COPII Vesicles revealed a coat built with polygonal units. The length of the side of the hexagonal/pentagonal units is close to the dimension of the central rod-like segment of Sec13/31. Partially uncoated profiles revealed strands of Sec13/31p stripped from the Vesicle surface. We conclude that the coat subunits form layers displaced from the membrane surface in reverse order of addition to the coat.

  • copii coated Vesicle formation reconstituted with purified coat proteins and chemically defined liposomes
    Cell, 1998
    Co-Authors: K Matsuoka, Lelio Orci, Mylene Amherdt, Sebastian Y Bednarek, Susan Hamamoto, Thomas Yeung
    Abstract:

    COPII Vesicle formation requires only three coat assembly subunits: Sar1p, Sec13/31p, and Sec23/24p. PI 4-phosphate or PI 4,5-bisphosphate is required for the binding of these proteins to liposomes. The GTP-bound form of Sar1p recruits Sec23/24p to the liposomes as well as to the ER membranes, and this Sar1p-Sec23/24p complex is required for the binding of Sec13/31p. Ultrastructural analysis shows that the binding of COPII coat proteins to liposomes results in coated patches, coated buds, and coated Vesicles of 50-90 nm in diameter. Budding proceeds without rupture of the donor liposome or Vesicle product. These observations suggest that the assembly of the COPII coat on the ER occurs by a sequential binding of coat proteins to specific lipids and that this assembly promotes the budding of COPII-coated Vesicles.

John E. Heuser - One of the best experts on this subject based on the ideXlab platform.

  • surface structure of the copii coated Vesicle
    Proceedings of the National Academy of Sciences of the United States of America, 2001
    Co-Authors: K Matsuoka, Lelio Orci, John E. Heuser
    Abstract:

    The spatial arrangement of COPII coat protein subunits was analyzed by crosslinking to an artificial membrane surface and by electron microscopy of coat proteins and coated Vesicle surfaces. The efficiency of COPII subunit crosslinking to phospholipids declined in order of protein recruitment to the coat: Sar1p > Sec23/24p ≫ Sec13/31p. Deep-etch rotary shadowing and electron microscopy were used to explore the COPII subunit structure with isolated proteins and coated Vesicles. Sec23/24 resembles a bow tie, and Sec13/31p contains terminal bilobed globular structures bordering a central rod. The surface structure of COPII Vesicles revealed a coat built with polygonal units. The length of the side of the hexagonal/pentagonal units is close to the dimension of the central rod-like segment of Sec13/31. Partially uncoated profiles revealed strands of Sec13/31p stripped from the Vesicle surface. We conclude that the coat subunits form layers displaced from the membrane surface in reverse order of addition to the coat.

Lelio Orci - One of the best experts on this subject based on the ideXlab platform.

  • surface structure of the copii coated Vesicle
    Proceedings of the National Academy of Sciences of the United States of America, 2001
    Co-Authors: K Matsuoka, Lelio Orci, John E. Heuser
    Abstract:

    The spatial arrangement of COPII coat protein subunits was analyzed by crosslinking to an artificial membrane surface and by electron microscopy of coat proteins and coated Vesicle surfaces. The efficiency of COPII subunit crosslinking to phospholipids declined in order of protein recruitment to the coat: Sar1p > Sec23/24p ≫ Sec13/31p. Deep-etch rotary shadowing and electron microscopy were used to explore the COPII subunit structure with isolated proteins and coated Vesicles. Sec23/24 resembles a bow tie, and Sec13/31p contains terminal bilobed globular structures bordering a central rod. The surface structure of COPII Vesicles revealed a coat built with polygonal units. The length of the side of the hexagonal/pentagonal units is close to the dimension of the central rod-like segment of Sec13/31. Partially uncoated profiles revealed strands of Sec13/31p stripped from the Vesicle surface. We conclude that the coat subunits form layers displaced from the membrane surface in reverse order of addition to the coat.

  • copii coated Vesicle formation reconstituted with purified coat proteins and chemically defined liposomes
    Cell, 1998
    Co-Authors: K Matsuoka, Lelio Orci, Mylene Amherdt, Sebastian Y Bednarek, Susan Hamamoto, Thomas Yeung
    Abstract:

    COPII Vesicle formation requires only three coat assembly subunits: Sar1p, Sec13/31p, and Sec23/24p. PI 4-phosphate or PI 4,5-bisphosphate is required for the binding of these proteins to liposomes. The GTP-bound form of Sar1p recruits Sec23/24p to the liposomes as well as to the ER membranes, and this Sar1p-Sec23/24p complex is required for the binding of Sec13/31p. Ultrastructural analysis shows that the binding of COPII coat proteins to liposomes results in coated patches, coated buds, and coated Vesicles of 50-90 nm in diameter. Budding proceeds without rupture of the donor liposome or Vesicle product. These observations suggest that the assembly of the COPII coat on the ER occurs by a sequential binding of coat proteins to specific lipids and that this assembly promotes the budding of COPII-coated Vesicles.

Thomas Yeung - One of the best experts on this subject based on the ideXlab platform.

  • copii coated Vesicle formation reconstituted with purified coat proteins and chemically defined liposomes
    Cell, 1998
    Co-Authors: K Matsuoka, Lelio Orci, Mylene Amherdt, Sebastian Y Bednarek, Susan Hamamoto, Thomas Yeung
    Abstract:

    COPII Vesicle formation requires only three coat assembly subunits: Sar1p, Sec13/31p, and Sec23/24p. PI 4-phosphate or PI 4,5-bisphosphate is required for the binding of these proteins to liposomes. The GTP-bound form of Sar1p recruits Sec23/24p to the liposomes as well as to the ER membranes, and this Sar1p-Sec23/24p complex is required for the binding of Sec13/31p. Ultrastructural analysis shows that the binding of COPII coat proteins to liposomes results in coated patches, coated buds, and coated Vesicles of 50-90 nm in diameter. Budding proceeds without rupture of the donor liposome or Vesicle product. These observations suggest that the assembly of the COPII coat on the ER occurs by a sequential binding of coat proteins to specific lipids and that this assembly promotes the budding of COPII-coated Vesicles.

Jurgen Denecke - One of the best experts on this subject based on the ideXlab platform.

  • secretory bulk flow of soluble proteins is efficient and copii dependent
    The Plant Cell, 2001
    Co-Authors: Belinda Phillipson, Ali Movafeghi, Peter Pimpl, Luis L P Dasilva, Andrew J Crofts, Philip J Taylor, David Robinson, Jurgen Denecke
    Abstract:

    COPII-coated Vesicles, first identified in yeast and later characterized in mammalian cells, mediate protein export from the endoplasmic reticulum (ER) to the Golgi apparatus within the secretory pathway. In these organisms, the mechanism of Vesicle formation is well understood, but the process of soluble cargo sorting has yet to be resolved. In plants, functional complements of the COPII-dependent protein traffic machinery were identified almost a decade ago, but the selectivity of the ER export process has been subject to considerable debate. To study the selectivity of COPII-dependent protein traffic in plants, we have developed an in vivo assay in which COPII Vesicle transport is disrupted at two distinct steps in the pathway. First, overexpression of the Sar1p-specific guanosine nucleotide exchange factor Sec12p was shown to result in the titration of the GTPase Sar1p, which is essential for COPII-coated Vesicle formation. A second method to disrupt COPII transport at a later step in the pathway was based on coexpression of a dominant negative mutant of Sar1p (H74L), which is thought to interfere with the uncoating and subsequent membrane fusion of the Vesicles because of the lack of GTPase activity. A quantitative assay to measure ER export under these conditions was achieved using the natural secretory protein barley α-amylase and a modified version carrying an ER retention motif. Most importantly, the manipulation of COPII transport in vivo using either of the two approaches allowed us to demonstrate that export of the ER resident protein calreticulin or the bulk flow marker phosphinothricin acetyl transferase is COPII dependent and occurs at a much higher rate than estimated previously. We also show that the instability of these proteins in post-ER compartments prevents the detection of the true rate of bulk flow using a standard secretion assay. The differences between the data on COPII transport obtained from these in vivo experiments and in vitro experiments conducted previously using yeast components are discussed.