Studies of Membrane Formation in Tetrahymena pyriformis

An experimental system has been developed to analyze the synthesis and intracellular deployment of proteins in growing cells of Tetrahymena pyriformis. There are marked differences in the rates at which radioactive proteins appear in various parts of the cell, including certain membranes, following [3H]leucine pulse-chase labeling experiments. Proteins labeled during a 5-min exposure to the isotope require approximately 2 hours for intracellular dissemination. Polyacrylamide gel electrophoresis indicated that all individual proteins of a particular cell fraction accumulate radioactivity at equivalent rates, except in the case of cilia. Further subfractionation of purified cilia yielded three components: matrix, axonemes, and membranes. The latter fraction was found to have a relatively simple protein composition involving only four major constituents. The matrix fraction, which had the highest specific radioactivity immediately following the 3H pulse, appeared to contain new proteins being conveyed to membranes and axonemes. By 2 hours following the chase, all three subfractions of the cilia had nearly equivalent specific radioactivities. Radioactive membrane lipids also seem to be transported into cilia via the matrix fraction. However, ciliary membrane lipids accumulate radioactivity more slowly than do the membrane proteins, apparently because of the tendency for radioactive lipids to exchange with pre-existing lipid molecules of membranes encountered during the first stages of intracellular transport. Other major differences in rates of lipid and protein labeling qualify Tetrahymena as a potentially rewarding model system for studying the process of membrane assembly.