Abstract
The mitochondrial unfoldase–protease complex CLPXP has been implicated in diverse mitochondrial pathways. A common feature of proposed CLPXP substrates is that their abundance or activity must be precisely regulated, as either excessive or insufficient activity can impair mitochondrial function. Heme biosynthesis exemplifies this principle: the heme biosynthetic enzyme ALAS is negatively regulated by heme through CLPXP-mediated degradation, and disruption of this regulatory pathway—including hyperactive ALAS variants or mutations that impair CLPX function—can cause erythropoietic protoporphyria. In Chapter 2, we describe the mechanism for heme-dependent degradation of ALAS by CLPXP and demonstrate that it requires a heme-sensitive adaptor protein, POLDIP2. We found that POLDIP2 and ALAS2 co-coordinate a single heme molecule, which serves as a molecular glue to recruit ALAS2 for CLPXP-mediated degradation beginning at the C terminus. This mechanism explains certain erythropoietic porphyria disease phenotypes that are caused by C-terminal truncation of the ALAS2 protein. In a model cell system for erythropoiesis, we validate that loss of POLDIP2 causes a similar phenotype to ALAS truncation variants that cause erythropoietic porphyria and measure similar accumulation of a toxic heme precursor.POLDIP2 is constitutively associated with CLPX, raising the question of how it recognizes substrates whose degradation does not rely on heme. In Chapter 3, we identify mitochondrial ribosome assembly factor ERAL1 as one such substrate. Although CLPXP promotes removal of ERAL1 from immature small subunit (SSU) complexes, ERAL1 is also essential for 12S rRNA stability and mitoribosome assembly. Thus, like ALAS, ERAL1 presents a regulatory challenge: sufficient ERAL1 must be maintained to support its physiological function, while excess or persistent ERAL1 must be eliminated. We describe a mechanism by which both CLPX and POLDIP2 can bind ERAL1, and this binding and degradation is inhibited when ERAL1 is bound to GTP or GDP. A missense mutation to the GTP binding pocket of ERAL1 causes a form of Perrault Syndrome which results in low basal levels of ERAL1 in the mitochondria. We found that this disease variant is degraded by CLPXP and its degradation is not inhibited by guanine nucleotide, likely because it cannot adequately bind GTP or GDP. As a result, the mutation may prevent ERAL1 from adopting the nucleotide-bound states that inhibit degradation, leading to constitutive degradation by CLPXP.
GTP binding is essential for ERAL1 stability, but an important question remains: what signal informs CLPXP and POLDIP2 that ERAL1 has completed its role in ribosome assembly and should be removed from the SSU? In Appendix I, we present preliminary studies of two SSU-associated proteins as candidate ERAL1 regulators. The first, RCC1L, is a putative guanine nucleotide exchange factor (GEF) that associates with SSU complexes alongside ERAL1. The second is RBFA, which replaces ERAL1 during maturation of the SSU. We investigate whether RBFA binding promotes ERAL1 release and subsequent recognition by the CLPXP–POLDIP2 degradation machinery.