To investigate the role of newly synthesized proteins during autophagic sequestration and degradation, the effects of protein synthesis inhibition on autophagic vac-

Classical or macroautophagy is a normal degradative pathway that exists in all eukaryotic cells (for reviews see Holtman, 1989; Seglen and Bohley, 1992). This degradative mechanism involves the sequestration of intracellular material, including organelles, by membranes of the ER (Marzella and Glaumann, 1987; Dunn, 1990a; Furuno et al., 1990; Ueno et al., 1991), to form a unique nascent autphagic vacuole (AV). The formation of nascent AVs is followed very rapidly by the delivery of lysosomal enzymes from pre-existing lysosomes to form degradative AVs (Ericsson, 1969; Lee et al., 1989; Lawrence and Brown, 1992). This autophagic process is the primary mechanism responsible for the turnover of unnecessary or dysfunctional organelles as well as for a random degradation of cytoplasmic proteins (Ahlberg et al., 1985; Ballard, 1987; Holtzman, 1989; Kopitz et al., 1990). Autophagy also plays a key role in embryonic programmed cell death, such as occurs during normal animal development (Holtzman, 1989; Clarke, 1990). Autophagy can be stimulated above this basal level by a variety of environmental stresses that enhance the need for cells to utilize their normal autophagic mechanism to survive. The complexity of this multi-step process suggests that it must be tightly regulated. The literature regarding whether or not autophagy requires protein synthesis is contradictory. On one hand, cycloheximide has been found to have no inhibitory effect on autophagy. For example, several groups have reported that pretreatment with cycloheximide did not inhibit the formation of glucagon-induced AVs or their conversion into a degradative compartment (Arstila and Trump, 1968; Shelburne et al. 1973; Wong and Woo, 1987). On the other hand, Amenta and Brocher (1981) reported that cycloheximide failed to inhibit autophagy stimulated by glucagon, but rapidly abrogated AV formation when the microtubule-disrupting compound vinblastine was used as a stimulus. Similarly, others have found that cycloheximide pretreatment decreased autophagic degradation or inhibited the formation of AVs in liver and pancreas of vinblastine-treated rats (Marzella and Glaumann, 1980a; Oliva et al., 1992). Because of the different experimental methods employed, it is very difficult to draw a final conclusion about the necessity of protein synthesis for autophagy. Two critical variables among these studies are the methods used to induce autophagy, and the timing of exposure to cycloheximide, including the length of treatment prior to the stimulation of autophagy. Studies using vinblastine to stimulate autophagy are particularly difficult to interpret due to its effect on microtubules. It has been proposed by some investigators that microtubule disruption inhibits the ability of 473 Journal of Cell Science 105,473-480 (1993) Printed in Great Britain © The Company of Biologists Limited 1993