**1. Introduction**

TCTP (Translationally Controlled Tumor Protein) is an evolutionarily-conserved and abundant protein among eukaryotic organisms. It is an essential protein for the development of multicellular organisms [1–3] and its main biological role is likely an anti-apoptotic activity [4–7]. However, it is also involved in many other core cell biological processes (reviewed in [8]). Despite it being a long time since its discovery in the 1980s [9] and subsequent intensive studies, the protein still remains a bit enigmatic, and new discoveries and new effects are still being described.

Recently, TCTP has also been found to affect autophagy [10,11]. Chen and colleagues reported that TCTP positively affects hypoxia and starvation-induced bulk non-selective autophagy [10]. On the other hand, Bae and colleagues have declared TCTP a negative regulator of basal and rapamycin-induced non-selective autophagy [11]. Since TCTP is an evolutionarily highly conserved protein, we used a pioneer model organism for studying autophagy, budding yeas<sup>t</sup> *Saccharomyces cerevisiae* [12], reviewed in [13–15] to test the effect of yeas<sup>t</sup> TCTP on autophagy. The *S. cerevisiae* is usually batch cultured and its growth in the culture is highly affected by a carbon source. When glucose is added to yeas<sup>t</sup> cells, they rapidly adapt to fermentation of the rich carbon source during a short

lag-phase. After the adaptation, they start to ferment the sugar and reach a maximal growth rate. This phase is called the exponential growth phase. Once glucose becomes limiting, yeas<sup>t</sup> cells enter a second lag-phase, known as a diauxic shift [16]. During the diauxic shift yeas<sup>t</sup> cells change their metabolism from fermentation to respiration. The diauxic shift is followed by a slow growing phase (post diauxic growth phase), during which ethanol, acetate, and other fermentation products are utilized by respiration. When the all carbon sources are exhausted yeas<sup>t</sup> cells enter a quiescence or stationary phase (G0) [16,17].

Yeast TCTP was originally described as a translation machinery associated protein, Tma19 [18]. Later, Tma19 was described as a microtubule and mitochondria interacting protein and renamed Mmi1 [19]. Therefore, we refer to the protein as Mmi1 hereafter. Mmi1 is a small 18.7 kDa, acidic (pI = 4.17), and highly abundant protein in exponentially growing yeas<sup>t</sup> cells corresponding to approximately 200,000 molecules per cell [20]. During the post-diauxic growth phase, Mmi1 is still a highly abundant protein exhibiting a steady-state level of expression [21], and its abundance continually decreases in the stationary phase [21,22]. Upon rapamycin treatment, the Mmi1 protein pool decreases [23], indicating that the Mmi1 expression in yeas<sup>t</sup> might be regulated by TOR pathway similarly to higher eukaryotic cells [24]. Further, the *mmi1* Δ strain exhibits a slow growth phenotype [19], indicating that Mmi1 is a pro-survival factor. Mmi1 is uniformly distributed in cytosol, but if stress is applied, its distribution is changed. Upon mild oxidative stress, Mmi1 translocates to mitochondria [25], while upon heat stress it relocalizes to the nucleus and mitochondria and is also present in stress granules [26]. Mmi1 role in the nucleus is not clarified yet. However, recently Bischof and colleagues suggested a model that the mitochondrial localization of the Mmi1/TCTP is responsible for the clearance of the mitochondrial membrane from harmful proteins in a time of stress [25], thereby protecting cells from apoptosis. Above the anti-apoptotic function, Mmi1 a ffects a wide range of biological functions and processes most likely through interaction with its binding partners. According to the BioGRID database [27], Mmi1 currently possess about 49 physically interacting protein partners. These proteins are mainly involved in cell cycle, transcription, translation, and protein degradation. Indeed, our previous results indicated that Mmi1 modulates activity of proteasomes [26], the major protein degradation system in all eukaryotic cells next to autophagy. Nevertheless, the e ffect of Mmi1 on autophagy in yeas<sup>t</sup> cells has not been tested yet. To test the question of whether Mmi1 a ffects non-selective autophagy, we induced autophagy through di fferent conditions and used independent approaches to monitor the autophagy.

Autophagy (here referred to macroautophagy) occurs constitutively at basal levels, but it is dramatically stimulated by starvation and by various stresses [28,29]. It allows cells to respond to various types of stresses and to adapt to changing nutrient conditions [30]. Autophagy can be either a non-selective self-consumption or a selective consumption of specific cargoes or organelles. The bulk autophagy is completely inhibited in nutrient-rich conditions, but can be induced by shifting cells to starvation medium [31] or by addition of rapamycin [32], a potent inhibitor of TORC1 (target of rapamycin complex 1) [33,34]. During non-selective autophagy a portion of cytosol is sequestered for degradation into double-membrane structures named autophagosomes, which are delivered to the vacuole and degradated by vacuolar hydrolases [35]. In *S. cerevisiae*, eighteen Atg proteins, Atg1–10, Atg12–14, Atg16–18, Atg29 and Atg31 play essential roles in autophagy, and these core proteins are required for the formation of autophagosomes (reviewed in [15,36]). When non-selective autophagy is induced, the Atg17-Atg29-Atg31 complex act as an essential sca ffold that facilitates formation of the preautosomal structure (PAS), from which the autophagosome is generated [37]. Autophagy is involved in a variety of physiological processes. In unicellular eukaryotes it takes care of cellular housekeeping and sustaining viability, and it is also essential for adaptation to a new host and formation of spores [38]. In higher eukaryotes it is important for cell survival and maintenance, and its dysfunction contributes to the pathologies of many diseases, e.g., cancer [39].

Here, we examined the e ffect of Mmi1 on bulk non-selective autophagy in yeast. Our results demonstrate a negative e ffect of Mmi1 on rapamycin-induced autophagy in contrast to nitrogen starvation-induced autophagy. Interestingly, the negative effect of Mmi1 on rapamycin-induced autophagy is detected after diauxic shift.
