Prohibitins have been implicated in diverse cellular processes ranging from cell proliferation, senescence, and the maintenance of mitochondrial morphology. Their activity on the molecular level, however, remained obscure. Our results reveal an unexpected role of prohibitins in the degradation of inner membrane proteins in mitochondria. A large complex containing the prohibitin homologues Phb1p and Phb2p of S. cerevisiae associates with the m-AAA protease. The prohibitin complex appears to modulate the activity of the protease, thus exerting a regulatory function during proteolysis.
A physical interaction of Phb1p and Phb2p with each other and with the m
-AAA protease was established by coimmunoprecipitation. Gel filtration analysis demonstrates that the m
-AAA protease subunits Yta10p and Yta12p are part of a supercomplex which contains the prohibitins and has a molecular mass greater than 2 MDa. This observation raises the possibility that Phb1p and Phb2p represent novel subunits of the m
-AAA protease. However, while proteolysis by the m
-AAA protease is required for the respiratory competence of the cells (2
), deletion of Phb1p or Phb2p does not cause any detectable growth phenotype (4
), demonstrating the activity of Yta10p and Yta12p in the absence of prohibitins. Consistently, the assembly of Yta10p and Yta12p is not affected in Δphb1
cells, nor is the formation of the Phb1p-Phb2p complex affected in cells lacking m
-AAA protease. Thus, in the inner membrane there are two large protein complexes, the prohibitin complex and the m
-AAA protease, which are formed independently but assemble with each other.
In mitochondria lacking Phb1p or Phb2p, proteolysis of nonassembled inner membrane proteins by the m
-AAA protease is enhanced. Although different effects on other substrate polypeptides cannot at this point be excluded, our findings indicate a negative regulatory role of the prohibitins in this process. The m
-AAA protease is most likely the direct target of prohibitin action. This inference is suggested by the physical association of prohibitins with the m
-AAA protease as well as by the apparent substrate specificity of prohibitin action. The turnover of Cox3p and Atp6p, both substrates of the m
-AAA protease (3
), was accelerated in the absence of prohibitins, whereas the rate of Cox2p degradation by the i
-AAA protease was not affected.
How could the prohibitin complex affect the proteolysis of membrane proteins by the m
-AAA protease? The large C-terminal domains of both Phb1p and Phb2p protrude into the intermembrane space, while Yta10p and Yta12p expose their catalytic sites to the matrix. This topology in the inner membrane suggests that the prohibitin complex exerts its effect via the membrane-embedded N-terminal part of the m
-AAA protease which includes segments exposed to the intermembrane space. Prohibitins may stabilize the m
-AAA protease in a conformation with lowered proteolytic activity. Alternatively, prohibitins may modulate the accessibility or conformation of membrane-embedded proteolytic substrates and thereby regulate the association of substrate polypeptides with the protease. By this means, the prohibitins could prevent the premature degradation of nonassembled membrane proteins by the m
-AAA protease and thereby ensure their proper assembly. Mitochondrial preproteins, which are present in a nonnative conformation during membrane translocation, might be protected against proteolytic attack in a similar manner. In mammalian cells, little prohibitin was found in association with cristae by immunoelectron microscopy, whereas it was enriched in the periphery of mitochondria (15
). Taking into account the biochemically established localization of prohibitins to the inner membrane (4
), this observation might point to an enrichment of prohibitins near the outer membrane, i.e., in the inner boundary membrane. A local inhibition of the m
-AAA protease by prohibitins in proximity to the import sites is therefore conceivable.
The role of prohibitins in the degradation of mitochondrial inner membrane proteins by the m
-AAA protease is reminiscent of previous findings in prokaryotes. The activity of the E. coli
AAA protease FtsH has been demonstrated to be negatively regulated by a complex of two homologous membrane proteins, HflK and HflC, which were found to interact directly with substrate polypeptides (16
). Interestingly, both HflK and HflC are distantly related to prohibitin family members of various organisms (Fig. ). Like Phb1p and Phb2p, HflK and HflC are anchored to the plasma membrane of E. coli
via an N-terminal membrane-spanning segment (17
). They expose a large domain to the periplasmic side of the plasma membrane of E. coli
, i.e., opposite that of FtsH (17
). The overall sequence identity of HflK and HflC to eukaryotic prohibitins, however, is significantly lower than within the prohibitin family. Nevertheless, the existence of distantly related proteins in bacteria and eukaryotes suggests that regulatory mechanisms for AAA proteases are conserved and derived from an early common ancestor.
FIG. 7 Sequence similarity of E. coli HflC with prohibitin family members. Amino acid sequences of S. cerevisiae Phb1p (ScPhb1p; P40961) and prohibitin from Arabidopsis thaliana (AtPhb; U69155) and Homo sapiens (hsPhb; P35232) were aligned with E. coli HflC (more ...)
The functional interaction of prohibitins with the m
-AAA protease raises the intriguing question of whether cellular activities previously attributed to prohibitins reflect their regulatory roles in membrane protein degradation. The observed requirement of prohibitins for efficient cell growth in the absence of the m
-AAA protease provides genetic evidence for a functional relationship of both complexes but cannot be explained by a physical interaction. Therefore, additional functions of prohibitins in mitochondria, likely linked to the degradation of inner membrane proteins and the activity of the m
-AAA protease, must exist. Prohibitins have recently been implicated in the regulation of mitochondrial morphology, as they were found to genetically interact with the mitochondrial inheritance components Mdm10p, Mdm12p, and Mmm1p in the outer membrane (4
). In view of our results, it is conceivable that this genetic interaction is caused by alterations in the turnover of inner membrane proteins in the absence of prohibitins. In contrast to cells lacking the i
-AAA protease subunit Yme1p (6
), however, evidence for defects in mitochondrial morphology in the absence of the m
-AAA protease, or after its overexpression, is lacking. Alternatively, the genetic interaction of prohibitins with mitochondrial inheritance components may reflect nonproteolytic functions of prohibitins. In view of the size of the prohibitin complex in the inner membrane, one can envision a scaffolding function of prohibitins which may allow for the assembly of a variety of mitochondrial proteins. Further insights into this question will require the identification of additional subunits of the prohibitin complex.
In view of the sequence conservation of prohibitins and AAA proteases from yeast to human (sequence identity of >50%), similar functions in all eukaryotic cells are likely. Another intriguing question raised by our findings is, therefore, how the effects of prohibitins on the m-AAA protease are linked to their roles in proliferation of mammalian cells and for cellular senescence. Alterations in mitochondrial physiology may change the energy level in the cell or its redox balance and thereby affect cell proliferation and aging. Prohibitins may affect mitochondrial activity by modulating the turnover of a short-lived regulatory protein by the m-AAA protease. It is therefore of interest to identify such putative substrates of the m-AAA protease and further characterize the specificity of prohibitin action during proteolysis.