1. Introduction {#sec1-cells-08-01231} =============== Autophagy is a conserved catabolic mechanism that regulates the turnover of cytoplasmic proteins and organelles \[[@B1-cells-08-01231],[@B2-cells-08-01231]\]. It is present in both healthy and diseased cells and is essential for maintaining cellular homeostasis \[[@B3-cells-08-01231],[@B4-cells-08-01231],[@B5-cells-08-01231]\]. Autophagy has been shown to prevent protein aggregation and toxicity, maintain protein homeostasis (proteostasis) and energy metabolism, as well as eliminate damaged and dangerous intracellular material (such as organelles and long-lived proteins) in order to limit cell death under various stress conditions \[[@B3-cells-08-01231]\]. During autophagy, portions of the cytoplasm are engulfed by a double membrane structure called an autophagosome, which is subsequently fused with lysosomes to form autolysosomes. The autophagosome is coated by a series of autophagy-related (Atg) proteins. The Atg5-Atg12 complex facilitates membrane nucleation and elongation, whereas the PI3K complex (Atg14L/Vps34) provides lipid-related molecules to elongate the phagophore \[[@B6-cells-08-01231],[@B7-cells-08-01231],[@B8-cells-08-01231]\]. Additional autophagy proteins facilitate membrane elongation, vesicle nucleation, and substrate recruitment, namely Atg9 and Atg16L. Atg9, which is a protein-free, single-pass membrane bound lipid, facilitates lipid delivery for membrane elongation. Atg16L binds to lipidated Atg9 and participates in autophagosome biogenesis by delivering membrane lipids to the growing autophagosome. Once autophagy has begun, it is a self-sustaining process. It continues until the cell is replenished with fresh supplies, such as free amino acids and ATP. This process is termed "autophagic flux". During autophagy, macromolecules and organelles are encapsulated into small membrane-bound vesicles called autophagosomes. The resulting structures fuse with the lysosome in order to form an autolysosome, where the cytoplasmic contents are ultimately degraded by lysosomal hydrolases. This process is known as "autophagic degradation", which is the central role of autophagy. The autophagy machinery can degrade various intracellular targets. The two most abundant molecular classes of autophagic substrates are soluble cytosolic proteins and long-lived proteins \[[@B9-cells-08-01231],[@B10-cells-08-01231]\]. The autophagic degradation pathway eliminates a portion of these two classes of targets by selective autophagic targeting and degradation. Several reports have shown that inhibition of autophagy can lead to the accumulation of p62/SQSTM1 protein, an autophagic receptor that regulates various cellular processes, including selective autophagy \[[@B11-cells-08-01231],[@B12-cells-08-01231]\]. p62/SQSTM1 contains an LC3-interacting region (LIR), which recognizes and selectively targets proteins to the autophagosome \[[@B13-cells-08-01231]\]. p62 binds to the autophagosome through LC3, a ubiquitin-like protein that is bound to the autophagosomal membrane through a lipid tail. It should be noted that, unlike LC3, p62 does not directly participate in the process of autophagosome nucleation or elongation. Instead, p62 promotes the recruitment of ubiquitinated protein aggregates to the expanding autophagosome. During starvation conditions, p62/SQSTM1 is recruited to the autophagosome through its LIR motif. This motif, which contains two aromatic residues on the side chains of amino acid tryptophan and phenylalanine (W^208^F^210^) \[[@B14-cells-08-01231]\], is present in several autophagy receptors, including p62/SQSTM1 \[[@B15-cells-08-01231]\], NBR1 \[[@B16-cells-08-01231]\], NDP52 \[[@B17-cells-08-01231]\], OPTN \[[@B18-cells-08-01231],[@B19-cells-08-01231]\], TAX1BP1 \[[@B20-cells-08-01231]\], and FUNDC1 \[[@B21-cells-08-01231]\]. Additionally, it has been shown that p62/SQSTM1 acts as a selective autophagy receptor for degradation of ubiquitinated protein aggregates through its LIR motif. In addition, autophagy-mediated degradation of p62/SQSTM1 can modulate the progression of various diseases \[[@B22-cells-08-01231]\]. Previous reports have shown that nutrient starvation triggers autophagy in cells \[[@B23-cells-08-01231],[@B24-cells-08-01231]\]. However, the mechanisms that control the activation of autophagy in response to nutrient deprivation are still not completely understood. Nutrient deprivation leads to the activation of TORC1 and TORC2 and, in response to nutrient deprivation, the activated TORC1 phosphorylates autophagy-related (ATG) proteins that promote autophagosome formation \[[@B3-cells-08-01231]\]. Previous reports have shown that, under nutrient starvation conditions, phosphorylation of TORC1 targets ULK1, Atg13, Atg17, Atg31, Atg32, and Atg34 \[[@B25-cells-08-01231],[@B26-cells-08-01231],[@B27-cells-08-01231],[@B28-cells-08-01231]\]. Phosphorylation of the Atg1 complex is another important regulatory mechanism that affects autophagy \[[@B29-cells-08-01231]\]. A large number of proteins, including Atg1, Atg6, Atg17, Atg29, Atg31, and Atg34 are phosphorylated by the protein kinase complex TORC1 \[[@B30-cells-08-01231]\]. ATG9 phosphorylation leads to the release of Atg9 from the inner membrane of the phagophore, which inhibits autophagosome biogenesis \[[@B31-cells-08-01231],[@B32-cells-08-01231],[@B33-cells-08-01231],[@B34-cells-08-01231],[@B35-cells-08-01231]\]. In particular, the phosphorylation of Atg9 by TORC1 in response to starvation is mediated by Atg14L and is a signal for inducing autophagy. Furthermore, TORC2 phosphorylates Atg9. This phosphorylation of Atg9 regulates the interaction between Atg9 and Atg16L, which is a regulator of autophagy \[[@B36-cells-08-01231],[@B37-cells-08-01231]\]. Previous reports have shown that autophagy is activated by starvation or growth factor withdrawal \[[@B31-cells-08-01231],[@B38-cells-08-01231]\]. These studies have focused on the functions of autophagy under extreme starvation conditions, such as starvation conditions. Previous reports have also shown that p62/SQSTM1 promotes selective autophagy, particularly targeting damaged mitochondria to autophagosomes \[[@B39-cells-08-01231],[@B40-cells-08-01231],[@B41-cells-08-01231],[@B42-cells-08-01231],[@B43-cells-08-01231]\]. Several studies have shown that p62/SQSTM1 is essential for selective autophagy during nutrient deprivation, suggesting that p62/SQSTM1 controls autophagosome formation and selective autophagy \[[@B22-cells-08-01231]\]. It is still unclear whether p62/SQSTM1 controls the selective autophagy of ubiquitinated protein aggregates in response to starvation conditions. Therefore, we investigated the role of p62/SQSTM1 as an autophagy receptor in starvation conditions. We observed that p62/SQSTM1 plays a role in the starvation-induced selective autophagy of ubiquitinated proteins and that the overexpression of p62/SQSTM1 can induce the accumulation of ubiquitinated protein aggregates. Therefore, this study reports a new mechanism, whereby p62/SQSTM1 mediates the degradation of ubiquitinated protein aggregates in starvation conditions. 2. Materials and Methods {#sec2-cells-08-01231} ======================== 2.1. Cell Culture {#sec2dot1-cells-08-01231} ----------------- Human colorectal cancer HCT116 cells were obtained from American Type Culture Collection (ATCC) (Manassas, VA, USA). Human cervical cancer HeLa cells were obtained from the Korean Cell Line Bank (Seoul, Korea). The p53-null cell line was generously donated by Dr. S. Han (Yonsei University, Seoul, Korea). The HCT116 and HeLa cells were maintained in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS), 1% penicillin/streptomycin, and 1% glutamine. The p53 wild-type cell line was maintained in DMEM medium supplemented with 10% FBS, 1% penicillin/streptomycin, and 1% glutamine. To induce autophagy, 10% FBS was replaced with FBS-free medium for 48 h. The culture medium was replaced every 24 h and then incubated for the indicated time periods. To treat cells with chloroquine (CQ), cells were incubated with 100 μM of chloroquine (Santa Cruz Biotechnology, Dallas, TX, USA) for 12 h. To induce serum starvation, cells were incubated in 0.5% FBS RPMI-1640