Cell-free assembly of rough and smooth endoplasmic reticulum

Smooth endoplasmic reticulum assembly was studied in a cell-free system using thin-section and freeze-fracture electron microscopy. Incubation of rat hepatocyte rough and smooth microsomes in the presence of ATP, GTP, cytosol (Xenopus egg) and an ATP-regenerating system led to assembly of membrane networks comprising a central core of interconnecting smooth tubules continuous with peripherally located rough membrane cisternae. Glucose-6-phosphatase cytochemistry confirmed the endoplasmic reticulum origin of the reconstituted membranes. When both ATP and GTP were omitted from the incubation medium, or when GTP was replaced by a variety of nucleotide analogues, including GTP gamma S, membrane aggregates contained only unfused microsomes. The presence of GTP alone stimulated assembly of rough membrane cisternae but had no effect on smooth membranes. Smooth tubule formation occurred independent of cytosol and an ATP-regenerating system, but did require GTP and ATP. Omission of ATP, or replacement of this nucleotide with a variety of analogues, including ATP gamma S, prevented tubule formation but did not affect the assembly of the rough membrane cisternae. Morphometric studies revealed sequential formation of rough membrane cisternae (0-60 minutes) followed by appearance of interconnecting smooth tubules (> 60 minutes). The amount of rough membrane cisternae per membrane network diminished with time after 60 minutes; that of smooth tubules increased. Thus GTP is required for reconstitution of rough membrane cisternae, both GTP and ATP are required for smooth tubule formation, and assembly of smooth tubules occurs as an outgrowth (i.e. via tubulation) from rough membranes.