Nucleoside diphosphate kinase B (NDKB) scaffolds endoplasmic reticulum membranes in vitro.
The mechanisms that structure the mammalian endoplasmic reticulum (ER) network are not fully understood. Here we show that salt extraction of semi-intact normal rat kidney (NRK) fibroblasts and subsequent incubation of the extracted cells with ATP resulted in dramatic ER network retraction. Under these conditions, addition of a single protein, Nucleoside Diphosphate Kinase B (NDKB), was sufficient to reverse the retraction and to promote ER network extension. The underlying mechanism of membrane extension involved direct lipid binding, as NDKB bound phosphatidylinositol (PtdIns)(4)P, PtdIns(4,5)P(2) and phosphatidic acid (PA); binding to these anionic lipids required clusters of basic residues on the surface of the NDKB hexamer; and amino acid changes in NDKB that blocked lipid binding also blocked ER network extension. Remarkably, purified NDKB transformed a uniform population of synthetic lipid vesicles into extensive membrane networks, and this also required its phospholipid-binding activity. Altogether these results identify a protein sufficient to scaffold extended membrane networks, and suggest a possible role for NDKB-like proteins, as well as phosphoinositides and/or acidic phospholipids, in modulating ER network morphogenesis.
Baughman
Cassandra
C
Program in Cell, Molecular and Developmental Biology, Tufts University School of Medicine, 136 Harrison Avenue, Boston, MA 02111, USA.
Morin-Leisk
Jeanne
J
Lee
Tina
T
D000818:Animals; D001681:Biological Assay; D004721:Endoplasmic Reticulum / Q000201:enzymology*; D007425:Intracellular Membranes / Q000201:enzymology*; D008081:Liposomes / Q000378:metabolism; D008958:Models, Molecular; D050505:Mutant Proteins / Q000378:metabolism; D054778:NM23 Nucleoside Diphosphate Kinases / Q000378:metabolism*; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000378:metabolism; D018129:Phosphatidylinositol Phosphates / Q000378:metabolism; D011485:Protein Binding; D051381:Rats
D008081:Liposomes; D050505:Mutant Proteins; D054778:NM23 Nucleoside Diphosphate Kinases; D019269:Phosphatidylinositol 4,5-Diphosphate; D018129:Phosphatidylinositol Phosphates; C037178:phosphatidylinositol 4-phosphate