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Requirement for Golgi-localized PI(4)P in fusion of COPII vesicles with Golgi compartments.

Lorente-Rodríguez A, Barlowe C - Mol. Biol. Cell (2010)

Bottom Line: The pleckstrin homology (PH) domain of human Fapp1, which binds phosphatidylinositol-4-phosphate (PI(4)P) specifically, was a strong and specific inhibitor of anterograde transport.Moreover, semi-intact cell membranes containing elevated levels of the ER-Golgi SNARE proteins and Sly1p were less sensitive to PI(4)P inhibitors.Finally, in vivo analyses of a pik1 mutant strain showed that inhibition of PI(4)P synthesis blocked anterograde transport from the ER to early Golgi compartments.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Dartmouth Medical School, Hanover, NH 03755, USA.

ABSTRACT
The role of specific membrane lipids in transport between endoplasmic reticulum (ER) and Golgi compartments is poorly understood. Using cell-free assays that measure stages in ER-to-Golgi transport, we screened a variety of enzyme inhibitors, lipid-modifying enzymes, and lipid ligands to investigate requirements in yeast. The pleckstrin homology (PH) domain of human Fapp1, which binds phosphatidylinositol-4-phosphate (PI(4)P) specifically, was a strong and specific inhibitor of anterograde transport. Analysis of wild type and mutant PH domain proteins in addition to recombinant versions of the Sac1p phosphoinositide-phosphatase indicated that PI(4)P was required on Golgi membranes for fusion with coat protein complex II (COPII) vesicles. PI(4)P inhibition did not prevent vesicle tethering but significantly reduced formation of soluble n-ethylmaleimide sensitive factor adaptor protein receptor (SNARE) complexes between vesicle and Golgi SNARE proteins. Moreover, semi-intact cell membranes containing elevated levels of the ER-Golgi SNARE proteins and Sly1p were less sensitive to PI(4)P inhibitors. Finally, in vivo analyses of a pik1 mutant strain showed that inhibition of PI(4)P synthesis blocked anterograde transport from the ER to early Golgi compartments. Together, the data presented here indicate that PI(4)P is required for the SNARE-dependent fusion stage of COPII vesicles with the Golgi complex.

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Characterization of membranes that overexpress anterograde ER-Golgi SNARE proteins and Sly1p. (A) Semi-intact cells from the overexpressor strain containing 2μ-BET1, 2μ-BOS1, 2μ-SEC22, 2μ-SED5, and 2μ-SLY1 (CBY3061) and the wild-type strain (CBY3062) were fractionated into soluble (S100) and pellet (P100) fractions for immunoblot analysis. (B) Budding reactions in which CBY3061 and CBY3062 microsomes were incubated in the absence (–) or presence (+) of COPII proteins for 30 min at 23°C. Immunoblots compare indicated proteins in budded vesicle fractions with one-tenth of total (T) budding reactions. Longer exposures (dark) are included for the Sec22p and Bet1p immunoblots.
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Figure 6: Characterization of membranes that overexpress anterograde ER-Golgi SNARE proteins and Sly1p. (A) Semi-intact cells from the overexpressor strain containing 2μ-BET1, 2μ-BOS1, 2μ-SEC22, 2μ-SED5, and 2μ-SLY1 (CBY3061) and the wild-type strain (CBY3062) were fractionated into soluble (S100) and pellet (P100) fractions for immunoblot analysis. (B) Budding reactions in which CBY3061 and CBY3062 microsomes were incubated in the absence (–) or presence (+) of COPII proteins for 30 min at 23°C. Immunoblots compare indicated proteins in budded vesicle fractions with one-tenth of total (T) budding reactions. Longer exposures (dark) are included for the Sec22p and Bet1p immunoblots.

Mentions: To determine fold overexpression and test whether overexpression influenced other ER-to-Golgi transport factors, we prepared semi-intact cell membranes from the strain overexpressing BET1, BOS1, SEC22, SED5, and SLY1 (from here on referred to as the overexpressor) for comparison with control strains. Experiments to assess the distribution of proteins contained in total, soluble, and membrane pellet fractions monitored the overexpressed proteins and a variety of other ER- and Golgi-localized markers (Figure 6A). We observed that Bet1p, Bos1p, Sec22p, Sed5p, and Sly1p were overexpressed three- to ninefold (compare total lanes), whereas the expression level and fractionation behavior of other marker proteins was not detectably altered.FIGURE 6:


Requirement for Golgi-localized PI(4)P in fusion of COPII vesicles with Golgi compartments.

Lorente-Rodríguez A, Barlowe C - Mol. Biol. Cell (2010)

Characterization of membranes that overexpress anterograde ER-Golgi SNARE proteins and Sly1p. (A) Semi-intact cells from the overexpressor strain containing 2μ-BET1, 2μ-BOS1, 2μ-SEC22, 2μ-SED5, and 2μ-SLY1 (CBY3061) and the wild-type strain (CBY3062) were fractionated into soluble (S100) and pellet (P100) fractions for immunoblot analysis. (B) Budding reactions in which CBY3061 and CBY3062 microsomes were incubated in the absence (–) or presence (+) of COPII proteins for 30 min at 23°C. Immunoblots compare indicated proteins in budded vesicle fractions with one-tenth of total (T) budding reactions. Longer exposures (dark) are included for the Sec22p and Bet1p immunoblots.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Figure 6: Characterization of membranes that overexpress anterograde ER-Golgi SNARE proteins and Sly1p. (A) Semi-intact cells from the overexpressor strain containing 2μ-BET1, 2μ-BOS1, 2μ-SEC22, 2μ-SED5, and 2μ-SLY1 (CBY3061) and the wild-type strain (CBY3062) were fractionated into soluble (S100) and pellet (P100) fractions for immunoblot analysis. (B) Budding reactions in which CBY3061 and CBY3062 microsomes were incubated in the absence (–) or presence (+) of COPII proteins for 30 min at 23°C. Immunoblots compare indicated proteins in budded vesicle fractions with one-tenth of total (T) budding reactions. Longer exposures (dark) are included for the Sec22p and Bet1p immunoblots.
Mentions: To determine fold overexpression and test whether overexpression influenced other ER-to-Golgi transport factors, we prepared semi-intact cell membranes from the strain overexpressing BET1, BOS1, SEC22, SED5, and SLY1 (from here on referred to as the overexpressor) for comparison with control strains. Experiments to assess the distribution of proteins contained in total, soluble, and membrane pellet fractions monitored the overexpressed proteins and a variety of other ER- and Golgi-localized markers (Figure 6A). We observed that Bet1p, Bos1p, Sec22p, Sed5p, and Sly1p were overexpressed three- to ninefold (compare total lanes), whereas the expression level and fractionation behavior of other marker proteins was not detectably altered.FIGURE 6:

Bottom Line: The pleckstrin homology (PH) domain of human Fapp1, which binds phosphatidylinositol-4-phosphate (PI(4)P) specifically, was a strong and specific inhibitor of anterograde transport.Moreover, semi-intact cell membranes containing elevated levels of the ER-Golgi SNARE proteins and Sly1p were less sensitive to PI(4)P inhibitors.Finally, in vivo analyses of a pik1 mutant strain showed that inhibition of PI(4)P synthesis blocked anterograde transport from the ER to early Golgi compartments.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Dartmouth Medical School, Hanover, NH 03755, USA.

ABSTRACT
The role of specific membrane lipids in transport between endoplasmic reticulum (ER) and Golgi compartments is poorly understood. Using cell-free assays that measure stages in ER-to-Golgi transport, we screened a variety of enzyme inhibitors, lipid-modifying enzymes, and lipid ligands to investigate requirements in yeast. The pleckstrin homology (PH) domain of human Fapp1, which binds phosphatidylinositol-4-phosphate (PI(4)P) specifically, was a strong and specific inhibitor of anterograde transport. Analysis of wild type and mutant PH domain proteins in addition to recombinant versions of the Sac1p phosphoinositide-phosphatase indicated that PI(4)P was required on Golgi membranes for fusion with coat protein complex II (COPII) vesicles. PI(4)P inhibition did not prevent vesicle tethering but significantly reduced formation of soluble n-ethylmaleimide sensitive factor adaptor protein receptor (SNARE) complexes between vesicle and Golgi SNARE proteins. Moreover, semi-intact cell membranes containing elevated levels of the ER-Golgi SNARE proteins and Sly1p were less sensitive to PI(4)P inhibitors. Finally, in vivo analyses of a pik1 mutant strain showed that inhibition of PI(4)P synthesis blocked anterograde transport from the ER to early Golgi compartments. Together, the data presented here indicate that PI(4)P is required for the SNARE-dependent fusion stage of COPII vesicles with the Golgi complex.

Show MeSH
Related in: MedlinePlus