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 fractions from the wall-less variant fz;sg;os-1V of Neurospora crassa reproduces in vitro translocation and processing of a secretory protein. The translation extract was isolated from the wall-less variant by gently lysing cells by a freeze-thaw procedure. This method yielded more extract 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 discontinuous sucrose gradients. The resultant microsomes were less inhibitory to translation of various transcripts and consisted of a more homogenous mixture of vesicles then microsomes prepared previously. Polyclonal antibodies directed against a polypeptide of approximately 75 kDa from the microsomes were used in indirect-immunofluorescence microscopy. The resultant fluorescent pattern shows a network of tubulo-reticular structures in a juxtanuclear region, which is the pattern expected of the rough endoplasmic reticulum.