New Insights Into Selective Autophagy in Yeast: Studies on Lap3

Cellular activities require the maintenance of balance between the synthesis and degradation of proteins. Regulation of protein degradation is less understood compared to protein synthesis. The ubiquitin-proteasome system contributes to the selective degradation of short-lived protein. Since most of cellular proteins have long lifetimes, the turnover of long-lived proteins is important to the understanding of cell physiology. Macroautophagy (hereafter simply referred to as autophagy) is an intracellular non-selective degradation system, which is well conserved in eukaryotes; autophagy transports cytoplasmic constituents to the lysosomes/vacuoles for degradation. The autophagic degradation is a cellular response to starvation and plays a role in recycling of cytoplasmic components, which is important for cellular remodeling, development, and differentiation. Screens in the yeast Saccharomyces cerevisiae have led to the identification of 31 autophagy-related (ATG)genes involved in autophagy. Much progress has been made in the functional analysis of these genes. Autophagy is initiated by the sequestration of cytoplasmic constituents in a double-membrane structure, termed the autophagosome. Fusion of an autophagosome membrane with the vacuole membrane results in the delivery of an inner vesicle (i.e., autophagic body). Eighteen Atg proteins comprise the core machinery essential for the biogenesis of the autophagosomes. Immuno-electron microscopy has revealed that ribosomes and typical cytosolic marker enzymes are present in the autophagosomes and autophagic bodies at the same densities as in the cytosol, indicating that autophagy is a non-selective and bulk degradation. If degradation of long-lived proteins is exclusively mediated by autophagy, all proteins might be expected to have similar lifetimes. Long-lived proteins, however, have a variety of lifetimes; therefore, the autophagic process would have some selectivity. Recently, different molecules or complexes are selectively recognized and delivered to the vacuoles via autophagy. Onodera and Ohsumi have reported that Ald6 is preferentially sequestered in autophagosomes and is degraded in vacuoles during prolonged starvation; this mechanism, however, is not understood well. Mechanisms of such cargo selection have been well studied for aminopeptidase I (Ape1), a vacuole-resident enzyme. Ape1 self-assembles and then forms an aggregate-like structure. Ape1 then is selectively incorporated into autophagosomes, and is transported into the vacuole during nitrogen starvation; this process is not degradation but biosynthesis. Thus, little is known about protein that is selectively degraded by yeast autophagy. To address the issue of selective degradation via autophagy, I focused on leucine aminopeptidase III (Lap3). Lap3 was originally isolated, along with Lap1/Ape2 and Lap4/Ape1, in a genetic screen and is a widely conserved cytoplasmic cysteine protease among eukaryotes. Lap3 self-assembles in the cytosol, and Lap3 also forms homohexameric complex. The first part of this study shows that Lap3 is a selective target of autophagy. When Lap3 tagged with GFP is overexpressed, it forms large aggregates next to the vacuole. Lap3 is transported to the vacuole in a manner dependent on autophagy during nitrogen starvation. Under these conditions, the rate of Lap3 transport is much higher than that of general cytosolic proteins; 27% of ［35S］Pho8△60, an indicator of general cytosolic proteins, is transported to the vacuole within 6 h, whereas approximately 50% of the Lap3 is transported to the vacuole in 1.5 h. These results show that Lap3 is selectively transported to the vacuole. I also identified that ATG11 is involved in Lap3 transport. ATG11 is essential for selective types of autophagy: selective degradation of peroxisomes (pexophagy) and mitochondria (mitophagy). Lap3 is apparently reduced after transports to the vacuole at 3 h. I hypothesized that the reduction in Lap3 was a result of degradation in the vacuole. To examine this possibility, the kinetics of Lap3 degradation via autophagic process was measured using temperature-sensitive (atg1ts) cell, which shows that most Lap3 is degraded in the vacuole within a couple of hours. Taken together, Lap3 is a novel target of selective degradation mediated by autophagy during nitrogen starvation. The yeast has a unique system, named the cytoplasm-to-vacuole-targeting (Cvt) pathway.This pathway utilizes common molecular machinery with autophagy under nutrient-rich and fermentable conditions, and constitutively delivers two vacuole-resident enzymes, Ape1 and α-mannosidase (Ams1), to the vacuole via a double-membrane structure (i.e., Cvt vesicle). The latter half of this study shows that the Cvt pathway is involved in not only a biosynthetic process but also the constitutive protein degradation. The Cvt pathway is enhanced during growth conditions in a medium containing glycerol as non-fermentable carbon source (YPGly). Under the conditions, the amount of vacuole-resident enzymes is increased; the lytic function in the vacuole may be important during YPGly growth, whereas non-selective autophagy is not detectable. I also found that in vacuolar protease-deficient pep4△) cells, single-membrane vesicles accumulate in the vacuole under the conditions. The intravacuolar vesicles are detected in ATG7-dependent manner, and ribosomes and membranes are apparently excluded from these vesicles. To investigate the content of the vesicle, I isolated vacuoles from pep4△ cells with or without ATG7 and subjected to proteomic analysis, leading to the identification of Lap3 and Ape1. When Lap3 tagged with GFP is endogenously expressed, it forms aggregates next to the vacuole during YPGly growth. Lap3 co-localizes with Ape1 and is transported into the vacuole. This transport requires ATG11 and ATG19, which are essential for the Cvt pathway. Atg19, which is a cargo receptor for Ape1, is immunoprecipitated with Lap3; Atg19 is likely to function as a receptor for Lap3 transport. These results show that Lap3 is constitutively transport to the vacuole during vegetative growth and is selectively sequestered in the Cvt vesicles. I assumed that Lap3 would be unstable in the vacuole, since Lap3 is the target of selective autophagy. To test this assumption, the stability of Lap3 in the vacuole was examined in atg1ts cells. The amount of Lap3 in the vacuole decreased to 50% of its initial level within 1.5 h. I also isolated vacuoles from cells with or without PEP4 and subjected to immunoblot, resulting that Lap3 is not detected in wild-type cell; Lap3 is degraded in the vacuole. Thus, I conclude that the Cvt pathway can function to eliminate certain proteins during vegetative growth. In this study, I revealed the follow facts; 1) Lap3 is a novel cargo of selective autophagy during nitrogen starvation, and 2) the Cvt pathway plays a role in protein degradation under growth conditions. Recently, protein degradation via autophagic process is thought to play a crucial role in elimination of aberrant protein complex, and this process would be performed constitutively and selectively. For instance, polyubiquitinated aggregates are recognized by p62, selectively sequestered by autophagosomes and are degraded in lysosomes. In contrast to p62, I do not know yet whether Lap3 are harmful or disadvantageous for yeast cells. As Lap3 is conserved widely in eukaryotes, it may serve as a model protein for analysis of selective autophagy in other organisms as well. Autophagy has traditionally been described as a non-selective degradation process. Several results shown in this thesis is the first study reporting that autophagy can be involved in selective and constitutive protein degradation in yeast cells. This thesis will allow researchers in this field to make new discoveries regarding the regulation and mechanisms of selective and constitutive autophagy