Particle-bound phytochrome: Association with a ribonucleoprotein fraction from Cucurbita pepo L.

Summary In the absence of ethylenediaminetetraacetic acid (EDTA) and added Mg²⁺, the phytochrome, RNA, protein, cytochrome c oxidase and NADPH-cytochrome c reductase in 20000 x g pellets from hypocotyl hooks of red-irradiated Cucurbita seedlings are more or less coincident in a single, broad band on linear sucrose gradients. The inclusion of 3 mM EDTA in the extraction, resuspension and gradient media has three major effects: (a) The phytochrome profile splits into two main bands; (b) the main RNA population shifts to a sharp peak which co-sediments with the lighter phytochrome band at 31S; (c) the main NADPH-cytochrome c reductase peak shifts to a lower density. This indicates that the EDTA dissociates a rough-endoplasmic-reticulum fraction into separate membrane and ribonucleoprotein (RNP) components, and that part of the phytochrome is associated with the latter. The 31S RNP fraction is 35–40% RNA, has a 260/235 nm absorption ratio of 1.36 and the RNA dissociates into small fragments in sodium dodecyl sulfate. More than 90% of the phytochrome and RNA in the isolated 31S fraction becomes pelletable upon the addition of 10 mM Mg²⁺. Higher Mg²⁺ levels release the phytochrome and some of the other protein present from the RNA which remains pelletable. The data indicate that the 31S RNP fraction may be degraded ribosomal material with extraneously bound protein, including phytochrome. Several aspects of phytochrome-binding to particulate fractions which have been reported in the literature are consistent with an interaction of P_fr with ribosomal material—degraded or otherwise.Abbreviations EDTA ethylenediaminetetraacetic acid - ER endoplasmic reticulum - P_fr far-red-absorbing form of phytochrome - P_r red-absorbing form of phytochrome - RNase ribonuclease - RNP ribonucleoprotein - SDS sodium dodecyl sulfate