The main mechanism for mitochondrial clearance is mitophagy, a selective type of autophagy that allows the degradation of damaged mitochondria. The importance of this process is highlighted by knowing that an impairment in mitochondrial function triggers an increase in reactive oxygen species production, which can in turn cause damage to cellular components (proteins, nucleic acid, and lipids) and trigger cell death (Lee et al., 2012).

During regular autophagy processes, stress or nutrient deprivation activates APM-activated protein kinase (AMPK), triggering two ubiquitin-dependent pathways (Figure 1A). One of these allows the assembly of the phagophore and involves several autophagy-related proteins (Atg), such as Atg5 and Atg7. The other aims to activate and lipidate LC3 (MAPLC3, microtubule-associated protein 1 light channel 3) by Atg4, a redox regulated protein. Atg4 and Atg7 cooperate to conjugate LC3 onto phosphatidylethanolamine in the lipid bilayer of the membrane originated from the ER-mitochondria contact site (Tooze and Yoshimori, 2010; Hamasaki et al., 2013). The elongated phagophore is then recruited to engulf targets via adaptor proteins, containing an LC3-interacting region (LIR) that forms a double-membrane autophagosome, which will fuse with a lysosome, initiating the degradation of the autophagosome components.

Upon mitochondria damage or depolarization, the mitochondrial membrane proteins are exposed and act as a beacon to recruit the phagophore membranes (Liu et al., 2014). An example is the PINK1 (P-TEN-induced kinase 1)-dependent recruitment of Parkin. Upon mitochondria depolarization, PINK1 accumulates at the OMM (outer mitochondrial membrane) and induces the mitochondrial translocation of Parkin, an RBR (ring-in-between)-type E3 ubiquitin ligase by direct phosphorylation (Kim et al., 2008; Narendra et al., 2010). The stabilization of Parkin at the OMM leads to the poly-ubiquitination of many proteins, inducing mitochondria fission and mobility stop and the phagophore recruitment by interacting with p62/SQSTM1, a LIR containing protein (Geisler et al., 2010). Unlike regular mitophagy induction, targeted mitochondria, during erythroblast maturation, are fully functional. BNIP3L/NIX, a BH3-only integral OMM protein first identified in mouse reticulocytes, appears to be the major mitochondrial protein involved during terminal differentiation (Schweers et al., 2007; Sandoval et al., 2008). This protein is upregulated during erythropoiesis and induces mitochondrial membrane depolarization and membrane conjugated LC3 recruitment to the mitochondria (Aerbajinai et al., 2003; Novak et al., 2010). Nix action is not mediated by its BH3 domain but rather seems to be due to a cytoplasmic short linear motif, acting as a cellular signal to recruit other proteins (Zhang et al., 2012). However, whether Nix-induced mitochondrial depolarization activates the Parkin-dependent pathway is still unknown (Yuan et al., 2017).

Recently, other mitochondrial receptors were found to participate in mitophagy, such as FUNDC1, induced by MARCH5, an E3 ubiquitin ligase acting in hypoxic condition (Chen et al., 2017), Bcl2-L-13 (Murakawa et al., 2015), optineurin (Wong and Holzbaur, 2014), and Prohibitin 2 (Wei et al., 2017). It remains unknown whether they play a role in erythroid maturation.

Canonical Atg proteins also participate in terminal maturation. In human erythropoiesis, LC3 cleavage is under the control of the endopeptidase Atg4 and is needed for autophagosome maturation (Betin et al., 2013). In mice, Ulk1 (Atg1) expression correlates with terminal differentiation and participates in mitochondria and ribosome elimination (Chan et al., 2007; Kundu et al., 2008). The ubiquitination-dependent pathway also plays a role in reticulocyte maturation but is not essential. Indeed, in Atg7^−/− reticulocytes, mitochondrial clearance is only partially affected (Zhang and Ney, 2009; Zhang et al., 2009). However, Nix and Ulk1 activation appears to be essential (Mortensen et al., 2010; Honda et al., 2014), suggesting the coexistence of both Atg5/Atg7-dependent and independent pathways during terminal differentiation.

Some studies suggest that the Atg5/7-independent degradation of mitochondria involves endosomal trafficking regulatory Rab proteins. Autophagosomes, formed in a Ulk1-dependent pathway, fuse with Golgi-derived vesicles and late endosomes in a Rab9a-dependent manner before they are targeted to the lysosomes (Wang et al., 2016). Interestingly, Rab proteins were also recently shown to be involved in mitochondria removal in a complete autophagy-independent pathway. Depolarized mitochondria appear to be engulfed in Rab5-positive endosomes that mature into Rab7-positive late endosomes and then fuse with lysosomes (Hammerling et al., 2017a,b). Unlike canonical autophagy, which involves the surrounding of a ubiquitin-decorated target by a double membrane structure, the entire mitochondria appears to be engulfed by an early endosome membrane invagination through the ESCRT machinery. Whether this might also occur in maturing erythroblasts is not known.

Mitophagy also appears to be transcriptionally regulated. Indeed, hemin-dependent differentiation of an erythroid cell line shows features of mitophagy (Fader et al., 2016). The NF-E2 transcription factor involved in globin gene expression also regulates mitophagy through the regulation of Nix and Ulk1 genes (Gothwal et al., 2016; Lupo et al., 2016). Another key regulator is the KRAB/KAP1-miRNA regulatory cascade, which acts as an indirect repressor of mitophagy genes in mice as well as in human cells, probably by the down and up regulation of a series of miRNAs, such as miR-351 that targets Nix (Barde et al., 2013).

In parallel to the autophagic pathway, cytosolic degradation seems to occur during reticulocyte maturation. 15-lipoxygenase (15-LOX), an enzyme that catalyzes the dioxygenation of polyunsaturated fatty acids, is translationally inhibited until the reticulocyte stage and acts to permeabilize organelle membranes, allowing proteasome access and degradation. Interestingly, only mitochondria elimination is affected, while ribosome clearance remains efficient when using a lipoxygenase inhibitor (Grüllich et al., 2001). This mechanism is still controversial, as 15-LOX might also act in the autophagy pathway as an OMM pH gradient disruptor that can induce mitophagy (Vijayvergiya et al., 2004), and on the oxidation of phospholipids conjugating with LC3 during the autophagosome formation; even so, these features, as shown in Table 1 were not demonstrated in erythroid cells yet (Morgan et al., 2015).

In general, autophagy plays an essential role in the elimination of other organelles, such as lysosomes, peroxisomes and ER. However, the literature presents only very few studies in erythroid cells (Table 1).

While Nix is required for mitochondria removal, Ulk1 is involved in ribosome and mitochondria degradation (Schweers et al., 2007; Kundu et al., 2008; Sandoval et al., 2008). Similarly, an efficient clearance of ribosomes and ER and the inhibition of mitophagy was observed in Atg7^−/− mice (Mortensen et al., 2010). These data suggest that non-autophagic or Atg7-independent autophagic pathways might exist for the elimination of other organelles (Figure 1A).

In non-erythroid cells from mammals, it was proposed that peroxisomes are eliminated by three different pathways: macroautophagy (Iwata, 2006), 15-LOX mediated (Yokota et al., 2001) and the peroxisomal Lon proteases (Yokota et al., 2008). Furthermore, the autophagic degradation of lysosomes (lysophagy) was recently identified in HeLa cells where it is mediated by ubiquitination and involves p62 protein (Hung et al., 2013). The similarities between pexophagy/lysophagy and mitophagy in non-erythroid cells suggest that autophagy pathways might also be involved in erythroblast terminal maturation.

After enucleation, reticulocytes mature in the bone marrow (R1) and then exit in the blood stream (R2) to complete the process. While the degradation of organelles starts at the time of enucleation, the elimination of mRNA occurs in the blood stream and is mediated by ribonucleases, generating nucleotides that are degraded by the erythroid pyrimidine nucleotidase. This elimination is crucial, as the deficiency in this enzyme causes hemolytic anemia (Valentine et al., 1974). mRNAs in R2 reticulocytes mainly belong to three overlapping categories: transport, metabolic and signal transduction (Lee et al., 2014), and their presence is essential to reach the mature RBC stage. This supports the importance of the exosome pathway for the final maturation into RBCs with an active elimination of other subcellular components.

Exosomes are small vesicles that are secreted into the extracellular medium from various kind of cells. PM invaginations form early endosomes that engulf various targets forming multivesicular bodies (MVB, late endosomes) that eventually fuse with the PM and release exosomes. In reticulocytes, this pathway is thought to be involved in cell volume and membrane remodeling to reduce volume and remove unwanted membrane proteins. This was first discovered in sheep reticulocytes where transferrin receptor (TfR) is first internalized into small vesicles of 100–200 nm before being engulfed into the MVBs (Pan et al., 1985; Johnstone et al., 1989). The internalization step is clathrin-dependent, and the degradation is lysosome-independent and occurs by exocytosis after the fusion of the MVBs with the PM as shown in Figure 1B (Killisch et al., 1992). This process is required for the final elimination of other membrane proteins that are essential for the reticulocyte but are absent in the mature cell. Proteins such as aquaporin-1 (AQP1) (Blanc et al., 2009), α4β1 integrin (Rieu et al., 2000), glucose transporter and acetylcholinestarase (Johnstone et al., 1987) are found in glycophorine-A (GPA) positive endosomes while cytoskeletal proteins, such as actin or spectrin have never been found in these endosomes (Liu et al., 2010).

While plenty of evidence notes the role of autophagy in removing organelles during terminal maturation, the degradation step itself shows discrepancies with canonical proteolysis involving lysosomal proteins because of the disappearance of the lysosomal compartment during the maturation and removal of LAMP2 by exocytosis (Barres et al., 2010). Recently, GPA-positive endosomes were found to express LC3 at the endosome membrane, suggesting the cooperation of both autophagy and exocytosis in the removal of remnant organelles in R2 reticulocytes. These hybrid vesicles contain mitochondria, Golgi and lysosomes might be formed by the fusion of the outer-membrane of the autophagosome and the PM derived endosome (Griffiths et al., 2012). The exocytosis of this vesicle might be favored by the spleen, as splenectomized patients present large vacuoles inside reticulocytes (Holroyde and Gardner, 1970).

It should be pointed out the importance of lipids domain such as cholesterol and sphingomyelin-enriched domains in the PM remodeling, as they were find both in membrane vesiculation specific sites (Leonard et al., 2017).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.