Abstract
Heme is an essential co-factor for a variety of cellular processes such as oxidative phosphorylation and transportation of molecular oxygen by hemoglobin. The biosynthesis of heme must therefore be tightly regulated to support these processes without depleting cellular iron or oversupplying toxic heme precursors. Dysregulation of heme biosynthesis can lead to disorders such as anemia and protoporphyria. The first and rate limiting step of heme biosynthesis is the production of aminolevulinic acid (ALA) by ALA synthase (ALAS) in the mitochondrial matrix. ALAS is activated through partial unfolding by the AAA+ mitochondrial unfoldase/protease pair CLPXP to reveal the binding site for the cofactor pyridoxal phosphate (PLP). CLPX, with its partner peptidase CLPP, has also been reported to fully unfold and degrade ALAS2 in heme replete conditions in vivo. However, the mechanism by which it bypasses the putative stop signal encountered during partial unfolding and activation has not been elucidated. Simple reconstitution of the CLPXP system with heme in vitro does not support degradation of ALAS2.In Chapter 2, we report the discovery that Polymerase Delta Interacting Protein 2 (POLDIP2) acts as an adaptor for mitochondrial CLPX to facilitate heme-dependent degradation of ALAS2 both in vitro and in vivo. As the name suggests, POLDIP2 was originally identified as an adaptor protein for Polymerase Delta in the nucleus wherein it helps overcome challenging DNA photodimers during DNA damage repair. POLDIP2 was recently identified as an interactor of mitochondrial CLPX, but with no ascribed function. We have determined that in this new role, POLDIP2 co-coordinates heme with the N-terminal extension of ALAS2 to form a stable complex and deliver ALAS2 for degradation by CLPXP but this extension is not required for degradation. Conversely, the C-terminal extension of ALAS2 is dispensable for complex formation but required for processive unfolding and degradation of ALAS2. Thus, POLDIP2 orchestrates the heme-dependent delivery and degradation of ALAS2 by mitochondrial CLPXP through these N- and C-terminal elements. These findings support a model of negative feedback regulation of heme biosynthesis as the accumulation of free heme in the cell would stabilize this complex, and promote the degradation of ALAS, thus downregulating heme supply. In Chapter 3, we discuss our preliminary efforts to biophysically characterize the determinants of heme dependent complex formation. There are two isoforms of ALAS, the housekeeping isoform ALAS1 and the erythroid isoform ALAS2. These isoforms share a high degree of sequence conservation within the catalytic core of these enzymes but are divergent in the N-terminal extension where the heme-coordination site is encoded. These determinants may vary between these two isozymes, thus tuning this feedback mechanism for different cell types. In Appendix I, we demonstrate nanomolar affinity between POLDIP2 and a CLPX hexamer through the use of a genetically encoded fluorophore.