In the present study we have identified the E1B 19-kD/ Bcl-2–binding protein Nip3 as a pro-apoptotic protein that localizes to mitochondria. Nip3 protein is unusual in several respects. It migrates on SDS-PAGE at a molecular mass that is much higher than the 21.54-kD predicted by the primary amino acid sequence and appears in vivo after transfection and in vitro after transcription and translation as 60- and 30-kD proteins. The 60-kD band does not dissociate after reduction or reduction and alkylation, nor after treatment with 6M urea (Chen, G., and A.H. Greenberg, unpublished data). Thus Nip3 appears to interact with a second protein very strongly and the evidence favors homodimerization. Three experiments support this conclusion. First, truncation of the COOH-terminal 31 amino acids results in the loss of the 60-kD Nip3 band and only a lower molecular weight protein is observed. Second, [35S]methionine-labeled tryptic peptide fragments of the 60-kD Nip3 were identical to the 28-kD Nip3163, and finally, Nip3 exhibits strong interaction with itself in the yeast two hybrid system. The inability to bind Nip3163 to interact with Nip3 or itself indicates that it cannot dimerize and is consistent with the requirement of the COOH-terminal portion of Nip3 for homodimerization.
The COOH-terminal domain of Nip3 not only influences dimer formation but also directs its expression to mitochondria. Nip3 colocalized with HSP60 which is primarily expressed in mitochondria (25
) while the mutant Nip3163
lacking the transmembrane domain was mostly expressed as a free cytosolic protein. A minor component of the Nip3163
protein was colocalized with the mitochondrial markers suggesting that it may interact by a mechanism other than membrane insertion, perhaps by binding to other mitochondrial proteins. Nip3163
does not interact with Nip3 or form a homodimer in vitro so it likely does not homodimerize with endogenous Nip3 in vivo.
Nip3 protein expression progressively decreases over time, which may reflect the increasing death of Nip3 expressing cells. Partial Bcl-2 suppression of Nip-3 induced apoptosis and a significant increase in Nip3 protein levels suggests that at least part of the Nip3 is lost by cell death. However, a progressive decrease in protein levels was also observed with the Nip3163
mutant which does not induce apoptosis, therefore another mechanism is affecting protein turnover. Nip3 contains PEST sequences suggesting that the protein may be susceptible to rapid degradation by proteases. PEST sequences commonly contain high local concentrations of amino acids P, E, S, T, and D flanked by charged amino acids (26
) and these are abundantly present in Nip3. Thus the posttranslational control of Nip3 expression through rapid protein degradation may constitute a mechanism for regulating the intracellular levels of a potentially lethal protein.
Nip3 was originally identified as an E1B 19K interacting protein, but the structure of Nip3 does not closely resemble other death agonists of the Bcl-2 family. Bcl-2 family agonists such as Bax act by heterodimerizing with anti-apoptotic family members and mutations in BH1 and BH2 domains in Bcl-2 or Bcl-XL
result in loss of binding and anti-apoptotic activity (27
). However, other studies suggest that pro- and anti-apoptotic Bcl-2 family members can act independently (28
) or through other family members such as BAD (29
). Nip3 does not contain either a BH1 or BH2 domain, although it may have a BH3 domain in which leucine110
, aspartic acid115
, and isoleucine117
are conserved based on the critical amino acids of the BH3 domain of BAK that determines BAK-Bcl-XL
). Other BH3 domain-only Bcl-2 family members that are death agonists have been described such as Bik/Nbk (32
), Bid (34
) and, more recently, Hrk (35
). Bik and Hrk have putative transmembrane domains while Bid is a cytoplasmic factor that is hypothesized to be recruited to membrane-associated Bcl-2 or Bax. Thus it is possible that Nip3 is a death factor of the Bcl-2 family related to Bik/Nbk, Bid, or Hrk.
Many Bcl-2 family members are integral membrane proteins that bear COOH-terminal regions that allow localization to the mitochondrial outer membrane, nuclear envelope and endoplasmic reticulum (15
). Localization of Bcl-2 to mitochondria also appears to be important in its suppression of cell death as mutants lacking the transmembrane domain are ineffective when overexpressed (14
). Nip3 shares the ability to localize to mitochondria and recent work suggests that mitochondria may play an important role in the regulation of apoptosis. They are necessary for the apoptotic activity of cytosolic extracts of Xenopus laevis
), and can initiate nuclear destruction when taken from cells undergoing apoptosis in in vitro reconstituted cell systems (16
). Nip3 localization in mitochondria places it in a position to influence mitochondrial function early in the apoptotic response. Nip3 can overcome Bcl-2 suppression of apoptosis so it might act on Bcl-2 or other anti-apoptotic family members that regulate mitochondrial permeability transition pores (16
) or cytochrome c release (18
). Nip3 preferentially forms homodimers even under reducing conditions and could provide a stable link between Bcl-2 homo- and/or heterodimers and other proteins perhaps forming larger complexes or altering their interaction with regulators such as BAD (30
In conclusion, we have identified the E1B 19K/Bcl-2 binding protein Nip3 as a homodimer localized to mitochondrial membrane. Nip3 expression in mitochondria induces apoptosis and can overcome Bcl-2 suppression of cell death, indicating that Nip3 is a novel pro-apoptotic protein.