A CELL-FREE TRANSLATION-TRANSLOCATION SYSTEM RECONSTITUTED WITH SUBCELLULAR-FRACTIONS FROM THE WALL-LESS VARIANT FZ-SG-OS-1V OF NEUROSPORA-CRASSA

A translation-translocation system reconstituted with subcellular frac tions from the wall-less variant fi;sg;os-1V of Neurospora crassa repr oduces in vitro translocation and processing of a secretory protein. T he translation extract was isolated from the wall-less variant by gent ly lysing cells by a freeze-thaw procedure. This method yielded more e xtract then the method developed previously (R. Addison, J. Biol. Chem . 262: 17031, 1987) as well as reducing microsomal contamination. The microsomal fraction was isolated from lysed cells using a series of di scontinuous sucrose gradients. The resultant microsomes were less inhi bitory to translation of various transcripts and consisted of a more h omogenous mixture of vesicles then microsomes prepared previously. Pol yclonal antibodies directed against a polypeptide of approximately 75 kDa from the microsomes were used in indirect-immunofluorescence micro scopy. The resultant fluorescent pattern shows a network of tubulo-ret icular structures in a juxtanuclear region, which is the pattern expec ted of the rough endoplasmic reticulum. (C) 1998 Academic Press.