R. Addison, A CELL-FREE TRANSLATION-TRANSLOCATION SYSTEM RECONSTITUTED WITH SUBCELLULAR-FRACTIONS FROM THE WALL-LESS VARIANT FZ-SG-OS-1V OF NEUROSPORA-CRASSA, Fungal genetics and biology (Print), 24(3), 1998, pp. 345-353
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.