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- Materials and Methods
Autophagy is a survival mechanism necessary for eukaryotic cells to overcome nutritionally challenged environments. When autophagy is triggered, cells degrade nonselectively engulfed cytosolic proteins and free ribosomes that are evenly distributed throughout the cytoplasm. The resulting pool of free amino acids is used to sustain processes crucial for survival. Here we characterize an autophagic degradation of the endoplasmic reticulum (ER) under starvation conditions in addition to cytosolic protein degradation. Golgi membrane protein was not engulfed by the autophagosome under the same conditions, indicating that the uptake of ER by autophagosome was the specific event. Although the ER exists in a network structure that is mutually connected and resides predominantly around the nucleus and beneath the plasma membrane, most of autophagosome engulfed ER. The extent of the ER uptake by autophagy was nearly identical to that of the soluble cytosolic proteins. This phenomenon was explained by the appearance of fragmented ER membrane structures in almost all autophagosomes. Furthermore, ER dynamism is required for this process: ER uptake by autophagosomes occurs in an actin-dependent manner.
Unicellular organisms such as yeast cells in nature are constantly challenged by changing external environments which force the cells to adapt their internal systems in order to survive. Nutritional starvation is the most common stress that yeast cells face throughout their lifetime. They are equipped with a multiplicity of mechanisms to overcome such stress by reorganizing their internal system (1,2). A well-studied aspect of such an adjustment is the tight regulation on expressing a specific group of genes. Genes required for protein synthesis and uptake of low affinity to specific amino acids are repressed under such conditions. Such tight regulation can be accomplished by adjusting transcription, down shifting translation and repressing ribosome biosynthesis (3).
Degradation of proteins is another important cell response against starvation stress. The vacuole is a major site for this degradation process. Expression of several groups of genes utilized in catabolism such as hydrolases is induced under such conditions (4). A frequently studied example of regulation by degradation is seen on a set of plasma membrane transporter proteins, tryptophan permease (Tat2p). Tat2p is endocytosed specifically under starvation conditions and degraded in the vacuole (5). In addition to regulated degradation of a specific group of proteins, bulk degradation of cytoplasmic constituents, called autophagy, takes place under starvation conditions. Autophagy is the process that nonselectively engulfs cytoplasm by forming a double membrane structure called autophagosome, which fuses with the vacuole for degradation (6). When autophagy is induced, 1–2% of cytoplasmic contents are degraded hourly and the resulting degradation products, amino acids, are reused to synthesize new proteins necessary for such conditions. Thus, autophagy largely contributes toward reorganizing cellular compositions to adapt to such stress.
In the course of studying the mechanistic aspects of autophagy, we recently found that an active flow of COPII vesicular traffic from the ER is required for autophagosome formation (7). Many studies have followed the changes in expression levels of proteins during the starvation, but what has not been studied is how the protein transport pathway, like the early secretory pathway from the ER, the protein production site of cell surface and secretory proteins, is affected. Here we characterize ER and Golgi resident proteins to see if and how the starvation stress modulates the early secretory pathway. Visualizing these proteins revealed dynamic changes in the early secretory pathway under starvation conditions. Significant amounts of both ER and Golgi proteins were transported to the vacuole during starvation, and ER proteins were degraded by autophagy.
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- Materials and Methods
In this study, we characterized the fate of the ER in response to starvation, and demonstrated a significant linkage to autophagy. Fluorescence microscopy showed that the lumenal ER marker, GFP-HDEL, was transported to the vacuole via autophagy during starvation. There is a possibility that translocation of ER proteins across the ER membrane might be inhibited under starvation conditions and such mistranslocated ER proteins are nonselectively engulfed by autophagosomes. We have checked the translocation of HDEL-GFP in sec61-1 mutant cells and the processed form but no precursor form was detected under starvation conditions by pulse-chase experiment (not shown). The possibility of mistranslocation can also be dismissed from the results of electron microscopic pictures; the pattern of gold-particles of HDEL-GFP in the cytoplasm was nowhere near the same as the pattern of ADH, a typical cytosolic marker protein (26). The gold particles of GFP-HDEL were not found as an evenly distributed pattern, with most of them being found on the peripheral and nuclear ER (Figure 5). They were also found inside autophagic bodies, and were detected on small-vesiculated structures. In addition, the ER membrane protein, Sec71p-GFP, was also transported to the vacuole via autophagy during starvation (Figure 3). A high concentration of DiOC6 that stains ER membrane, stained autophagic bodies accumulated in the vacuole. Furthermore, TEM pictures clearly showed the existence of rough ER in nearly all of autophagic bodies in carbon-starved cells (Figure 6). Although the total amount of ER delivered to the vacuole via autophagy is small, the frequency of ER in the autophagic bodies was strikingly high. We concluded that ER itself was engulfed and delivered to the vacuole via autophagy. Golgi proteins were also examined under starvation conditions. Although the delivery of some Golgi localized proteins to the vacuole was observed, such as that found with the ER, the process did not depend on autophagy.
Starvation-specific uptake of ER by autophagosome raises two interesting issues. The first is the existence of a fragmented form of the ER in the cytoplasm in addition to the nuclear and peripheral ER. ER in mammalian cells is transiently fragmented during the cell division cycle (27). In contrast, yeast ER is generally thought to maintain its integrity throughout the cell cycle. However, in addition to such generally accepted ER architecture, we have demonstrated the existence of ER fragments within the autophagosome. The simplest explanation for the localization of these ER fragments is that they exist in the cytoplasm and a small portion of that is nonselectively engulfed and transported to the vacuole by autophagy. However, immunoelectron microscopic observation barely detected ER fragments within the cytoplasm (Figure 5). Furthermore, after Lat-A treatment, which affects ER dynamics, the ER fragments were no longer engulfed by autophagosomes (Figure 7). This suggests that ER fragments are generated transiently as a result of the dynamic state of ER: ER is constantly branching and sliding the interconnected tubules of the mesh-like network (24,27), and such transient structures are captured by autophagosome and transported to the vacuole. The second interesting issue is the high frequency of ER in autophagosomes. Although autophagy is known to be a nonselective degradation system of cytoplasm, autophagic bodies scarcely engulfed glycogen granules, which are formed when cells are nitrogen starved and located rather near the peripheral ER within cells (Figure 5A). This leaves open the possibility that ER might be selectively engulfed by autophagosomes. A different and more plausible explanation is that an autophagosome forms near the ER, where transient ER fragments are constantly available, and it therefore nonselectively but highly efficiently engulfs these fragments. Autophagosomes are thought to form at or near a preautophagosomal structure (PAS), known to exist in the vicinity of the vacuole (12,28). Furthermore, our preliminary observations suggest that the PAS is mostly located at the nuclear vacuolar junction (unpublished data). We have recently reported that active flow from the ER is an important factor in forming the autophagosome (7). However, ER dynamism itself seems dispensable for autophagy since it was able to proceed in the presence of Lat-A. The linkage between ER and autophagosome formation highlighted by this and previous studies may provide a key to understanding the mechanism of autophagosome formation.
The ER and Golgi localized protein, invertase-Wbp1p, and GFP-HDEL behave differently in response to starvation. A fraction of GFP-HDEL was transported to the vacuole via autophagy, whereas transport of invertase-Wbp1p was dependent on the vacuolar protein sorting pathway. A previous report demonstrated that more than 90% of invertase-Wbp1p exists in the glycosylated Golgi form (19), meaning that it is efficiently packaged into COPII vesicles and leaves the ER. In contrast, the majority of DsRed-HDEL was localized in the ER and was not transported to the Golgi (Figure 1). A similar phenotype was observed with Kar2p: most of Kar2p remains in the ER and is not packaged into COPII vesicles, regardless of the presence of the HDEL retrieval signal (8). An idea that is becoming generally accepted is that the packaging of cargo proteins into COPII vesicles is an active process (29). Thus, we reasoned that the difference found between the two retrieval signals might be explained by differences in the efficiency of packing into COPII vesicles.
The finding that significant amounts of Rer1p and invertase-Wbp1p were transported to the vacuole upon induction of starvation (Figures 2 and 4) can be explained by regulation within the Golgi stacks. The Golgi apparatus consists of several compartments (cis, medial and trans) and is heavily regulated to maintain its dynamic compartments by a balance between anterograde and retrograde transports (30,31). This balance also determines the proper localization of each Golgi protein. It is possible that the retrieval system within the Golgi complex becomes less efficient during starvation; thus, the flow within the Golgi complex is no longer balanced and Golgi proteins are transported to the vacuole. However, the retrieval system was not completely abolished, as the Golgi-like distribution of Rer1p-GFP still existed even after a long starvation period. An alteration in COPI coats or COPI-related proteins might contribute to these changes as the localization of Rer1p and Wbp1p fusion protein is dependent on COPI vesicles (18,19). The further analysis on the efficiency of retrograde transport under starvation conditions remains an interesting proposition. We visualized Sec21p-GFP (γ-cop), one of the subunits of the COPI coat, but no significant changes in its distribution were found during starvation (not shown).
The changes observed in the secretory pathway could hold the following physiological significance. The delivery of ER and Golgi proteins to the vacuole may contribute to slowing down the secretory pathway by reducing levels of ER and Golgi. Another possibility is that this mechanism allows the cell to degrade and reuse proteins from the secretory pathway in addition to cytosolic proteins to generate the proteins required for survival. The starvation-specific ER and Golgi proteins transported to the vacuole were discovered in this study. Importantly, not all of the ER and Golgi was transported to the vacuole under these conditions. This is reasonable; the cells are only trying to survive starvation and will have to function normally as soon as they are no longer being starved. When yeast cells are placed in nutritionally challenging environments, many cellular functions change, including the cessation of growth and proliferation. It is likely that the transport of proteins to the plasma membrane is also down-regulated, as cells no longer need to expand their surface area. Further studies on the molecular mechanisms involved in these phenomena will elucidate the regulation of the secretory pathway during starvation.