In this article, we have characterized Apg7p as an Apg12p-activating enzyme and demonstrated its indispensable role in the yeast autophagy. Apg12p, a novel modifier protein, binds to Apg7p via a thioester bond. This binding requires both the active site cysteine (Cys507) and the ATP-binding domain. Accordingly, both the cysteine residue and the ATP-binding domain are essential for Apg12p–Apg5p conjugation and for autophagy as well. From these results, we conclude that Apg7p is a novel E1-like enzyme that activates Apg12p and is essential for autophagy.
Although we clearly showed the E1-like function of Apg7p, only the C-terminal region of Apg7p (residues 322 to 407) shows homology to Uba1p. According to the similarity boxes within other E1-like enzymes as described by Johnson et al. (1997)
and Liakopoulos et al. (1998)
, Apg7p has an ATP-binding domain within box I and an active site cysteine within box III that are essential for E1 function (Figure A); however, box III of Apg7p is not similar to those of Uba1p, Uba2p, or Uba3p. Also, neither similarity box II nor IV is present within Apg7p. Phylogenetic analysis of the regions containing box I and box III in the E1 enzymes also suggested that the relationship of Apg7p to the corresponding regions in UBA1, UBA2, and UBA3 family proteins is distant (Figure B) (Hatfield et al., 1990
; Handley et al., 1991
; McGrath et al., 1991
; Imai et al., 1992
; Dohmen et al., 1995
; Liakopoulos et al., 1998
; Mizushima et al., 1998a
; Osaka et al., 1998
; Yuan et al., 1998
). The comparison of the similarity boxes and phylogenetic analysis showed that Apg7p is distinct from other E1-like enzymes. It is of interest to know whether Apg7p functions as a heterodimer, because the Aos1p/Uba2p and Ula1p/Uba3p complexes function as heterodimers (del Olmo et al., 1997
; Johnson et al., 1997
; Liakopoulos et al., 1998
). At present, we have no answer.
Figure 8 Apg7p is distantly related to Uba1p and its relatives. (A) Schematic representation of the similarity domains among UBA1, UBA2, UBA3, and APG7. The putative active site cysteines are located within similarity box III (UBA domain), as indicated. The essential (more ...)
A working hypothesis of the Apg12p conjugation system consistent with these results is proposed in Figure . The C-terminal Gly of Apg12p is activated by Apg7p in an ATP-dependent manner. The Apg12p–Apg7p conjugate forms via the C-terminal Gly of Apg12p and Cys507
of Apg7p. The reaction probably occurs in the cytosol or the cytoplasmic side of an Apg12p-associated compartment(s), because Apg7p is mainly present in the cytoplasm (Figure ) (Kim et al., 1999
). By analogy to ubiquitination and related modifications, there may be novel E2 and E3 enzymes for the conjugation system. One such candidate for E2 is Apg10p, because the Apg12p–Apg5p conjugate is not observed in an apg10
mutant. Cloning of the APG10
gene and biochemical analysis will reveal the function of Apg10p. Screening other candidates will be performed using an Apg12p-affinity column, an Apg7p-affinity column, or two-hybrid screening.
Figure 9 Working hypothesis for the function of Apg7p in the Apg12p conjugation system. Apg12p associates with Apg7p. The C-terminal Gly of Apg12p conjugates with Cys507 in Apg7p via a thioester bond in an ATP-dependent manner. The reaction occurs in the cytoplasm (more ...)
It remains to be determined at which step in autophagy the Apg12p conjugation system works. Apg7p is mainly present in the cytosol, whereas more than half of Apg12p is localized in membrane compartment(s) or a large complex. The Apg12p–Apg7p binding probably occurs in the cytosol or the cytoplasmic side of an Apg12p-associated structure(s). Recently, Yuan et al. (1999)
suggested that a quite similar conjugation system is required for microautophagy in Pichia pastoris
. Microautophagy is the sequestration of cytoplasmic components (peroxisomes in this case) by an invagination of the vacuolar membrane. In a conjugation-deficient mutant (gsa7
), the sequestration is not accomplished completely, probably because of defects in the membrane fusion step. Gsa7p, a P. pastoris
homologue of Apg7p, was shown to be conjugated to a small protein through a thioester bond. Although a modifier and substrate(s) have not been identified, it strongly suggests that the Apg12p conjugation system is essential for microautophagy. Because the process of microautophagy is quite different from that of macroautophagy, it would be interesting if similar machinery is involved in both processes.
We have identified a human Apg12p homologue and have observed that the Apg12p homologue also conjugates with a human Apg5p homologue, which has been identified as an apoptosis-specific protein (Hammond et al. 1998
; Mizushima et al. 1998b
). A BLAST search of Apg7p in the Expressed Sequence Tag database showed potential mammalian homologues of Apg7p. Recently, an Apg7p homologue in P. pastoris
, Gsa7p, and a human Gsa7p/Apg7p homologue were identified by Yuan et al. (1999)
. The human Gsa7p/Apg7p homologue is expressed in various tissues as revealed by Northern analysis (Tanida and Kominami, unpublished observations). These findings suggest that the Apg12p conjugation system generally functions in eukaryotes. By analogy to the Apg5p homologue, an Apg7p homologue may play a significant role in the autophagic and apoptotic pathways in mammalian cells. Cloning and biochemical analyses of Apg7p homologues will reveal the function of the novel protein conjugation system in mammalian cells.
Apg7p is distantly related to UBA1, UBA2, and UBA3 family proteins (Figure A). Nevertheless, Apg7p functions as an E1 enzyme for Apg12p–Apg5p conjugation. These findings suggest that the ubiquitin-like protein modification system will form a ubiquitous regulatory system in eukaryotes, not specific to ubiquitin and ubiquitin-like proteins. Further analyses of potential E1 enzymes may reveal a novel regulatory system by posttranslational modification.