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. 2009 Dec;16(12):1622-9.
doi: 10.1038/cdd.2009.110. Epub 2009 Aug 14.

Hax1 lacks BH modules and is peripherally associated to heavy membranes: implications for Omi/HtrA2 and PARL activity in the regulation of mitochondrial stress and apoptosis

D V Jeyaraju  1 , G Cisbani, O M De Brito, E V Koonin, L Pellegrini
Affiliations

Hax1 lacks BH modules and is peripherally associated to heavy membranes: implications for Omi/HtrA2 and PARL activity in the regulation of mitochondrial stress and apoptosis

D V Jeyaraju et al. Cell Death Differ. 2009 Dec.

Abstract

Hax1 has an important role in immunodeficiency syndromes and apoptosis. A recent report (Chao et al., Nature, 2008) proposed that the Bcl-2-family-related protein, Hax1, suppresses apoptosis in lymphocytes and neurons through a mechanism that involves its association to the inner mitochondrial membrane rhomboid protease PARL, to proteolytically activate the serine protease Omi/HtrA2 and eliminate active Bax. This model implies that the control of cell-type sensitivity to pro-apoptotic stimuli is governed by the PARL/Hax1 complex in the mitochondria intermembrane space and, more generally, that Bcl-2-family-related proteins can control mitochondrial outer-membrane permeabilization from inside the mitochondrion. Further, it defines a novel, anti-apoptotic Opa1-independent pathway for PARL. In this study, we present evidence that, in vivo, the activity of Hax1 cannot be mechanistically coupled to PARL because the two proteins are confined in distinct cellular compartments and their interaction in vitro is an artifact. We also show by sequence analysis and secondary structure prediction that Hax1 is extremely unlikely to be a Bcl-2-family-related protein because it lacks Bcl-2 homology modules. These results indicate a different function and mechanism of Hax1 in apoptosis and re-opens the question of whether mammalian PARL, in addition to apoptosis, regulates mitochondrial stress response through Omi/HtrA2 processing.

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Figures

Figure 1
Figure 1. Hax1 does not contain BH1 or BH2 modules or a transmembrane domain
Multiple alignment of selected Hax1 sequences from diverse animals. The numbers between aligned blocks indicate poorly conserved sequence segments that are not shown. Secondary structure (SS) predicted with three methods is shown above the alignment: “c” indicates random coil (disordered structure), “h” indicates α-helix, and “e” indicates (β-strand (extended conformation). Amino acid residues that are conserved in all aligned sequences are shown in bold type, and the three invariant aspartates that comprise a putative metal-binding site are shown by reverse shading. The positions of purported BH1 and BH2 modules are shown above the respective regions of the alignment using the alignment from (22) for BH1, and arbitrarily centering the alignment on a conserved hydrophobic residue for BH2. “SS_BH” denotes the consensus secondary structure motifs of BH1 and BH2 modules derived from multiple crystal and NMR structures of Bcl-2-family proteins (24). The position of the transmembrane domain (TMD) predicted in (22), but not In our analysis, is indicated in the C-terminal block of the alignment.
Figure 2
Figure 2. Hax1 is not an integral membrane protein
Alkaline extraction of heavy membranes isolated from HeLa cells (200 μg); whereas membrane-associated proteins and proteins associated to membrane-bound proteins (e.g. UQCRC2) are solubilized in the supernatant, integral membrane proteins like PARL are recovered in the membrane pellet. Lack of Hax1 integration in heavy membranes is consistent with our computational analysis, which does not predict any potential transmembrane domain.
Figure 3
Figure 3. Hax1 is not detected in mitochondria
Immunoblot analysis of Percoll-purified fractions of mouse livers. In this preparations, lack of cross-contaminating organelles was assayed by electron microscopy (not shown); inner and outer mitochondria membrane integrity was tested for presence of diffusible proteins of the IMS (cytochrome c) and mitochondrial matrix (MnSOD); purity from membranes associated cytosolic proteins by absence of actin. The left panel shows that, upon normalization for cytohrome c and MnSOD, endogenous Hax1 is detected in heavy membranes but not purified mitochondria, a finding consistent with lack of Hax1 in the human and murine mitochondrial proteome (27). Note that association of Hax1 with heavy membranes, but not with ER-enriched light membranes (framed panel), suggests association of Hax1 to the cytosolic side of the outer mitochondrial membrane.
Figure 4
Figure 4. PARL does not have a domain that coordinates interaction to Hax1 in the IMS
a, schematic representation of the structure and domain composition of PARL (28, 30). b, co-immunoprecipitation of Hax1 with mutant forms of PARL in which the IMS domains (loop-A and C-terminus) have been deleted. Note that PACT formation in these mutants indicate that the proteins are correctly imported and folded in the inner membrane. c, mammalian two-hybrid assays fails to identify a domain in PARL that could mediate interaction to Hax1.
Figure 5
Figure 5. Hax1 binding to PARL is unspecific
a, in vitro reconstitution of PARL/Hax1 complex by mixing lysates of HEK293 cells transfected with a construct expressing PARL-Flag-CT or Hax-HA-CT. b, in vitro reconstitution of PARL/Hax1 complex by mixing lysates of heavy membranes (150 μg of proteins per genotype used) isolated from MEFs +/+, Hax1 -/-, or Parl -/- .
Figure 6
Figure 6. PARL proteolytic activity does not require Hax1
Genetic ablation of Hax1 does not impair the generation of PACT, a shorter form of PARL that requires PARL activity supplied in trans (33).
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