The ubiquitous mitochondrial protein unfoldase CLPX regulates erythroid heme synthesis by control of iron utilization and heme synthesis enzyme activation and turnover,

Apolipoprotein B-containing lipoproteins (B-lps) are essential for the transport of hydrophobic dietary and endogenous lipids through the circulation in vertebrates. Zebrafish embryos produce large numbers of B-lps in the yolk syncytial layer (YSL) to move lipids from yolk to growing tissues. Disruptions in B-lp production perturb yolk morphology, readily allowing for visual identification of mutants with altered B-lp metabolism. Here we report the discovery of a missense mutation in microsomal triglyceride transfer protein (Mtp), a protein that is essential for B-lp production. This mutation of a conserved glycine residue to valine (zebrafish G863V, human G865V) reduces B-lp production and results in yolk opacity due to aberrant accumulation of cytoplasmic lipid droplets in the YSL. However, this phenotype is milder than that of the previously reported L475P stalactite (stl) mutation. MTP transfers lipids, including triglycerides and phospholipids, to apolipoprotein B in the ER for B-lp assembly. In vitro lipid transfer assays reveal that while both MTP mutations eliminate triglyceride transfer activity, the G863V mutant protein unexpectedly retains~80% of phospholipid transfer activity. This residual phospholipid transfer activity of the G863V mttp mutant protein is sufficient to support the secretion of small B-lps, which prevents intestinal fat malabsorption and growth defects observed in the mttp stl/stl mutant zebrafish. Modeling based on the recent crystal structure of the heterodimeric human MTP complex suggests the G865V mutation may block triglyceride entry into the lipid-binding cavity. Together, these data argue that selective inhibition of MTP triglyceride transfer activity may be a feasible therapeutic approach to treat dyslipidemia and provide structural insight for drug design. These data also highlight the power of yolk transport studies to identify proteins critical for B-lp biology.

26Microsomal triglyceride transfer protein (MTP) transfers triglycerides and phospholipids and is 27 essential for the assembly of Apolipoprotein B (ApoB)-containing lipoproteins in the 28 endoplasmic reticulum. We have discovered a zebrafish mutant (mttp c655 ) expressing a C-29 terminal missense mutation (G863V) in Mttp, one of the two subunits of MTP, that is defective at 30 transferring triglycerides, but retains phospholipid transfer activity. Mutagenesis of the 31 conserved glycine in the human MTTP protein (G865V) also eliminates triglyceride but not 32 phospholipid transfer activity. The G863V mutation reduces the production and size of ApoB-33 containing lipoproteins in zebrafish embryos and results in the accumulation of cytoplasmic lipid 34 droplets in the yolk syncytial layer. However, mttp c655 mutants exhibit only mild intestinal lipid 35 malabsorption and normal growth as adults. In contrast, zebrafish mutants bearing the 36 previously identified mttp stl mutation (L475P) are deficient in transferring both triglycerides and 37 phospholipids and exhibit gross intestinal lipid accumulation and defective growth. Thus, the 38 G863V point mutation provides the first evidence that the triglyceride and phospholipid transfer 39 functions of a vertebrate MTP protein can be separated, arguing that selective inhibition of the 40 triglyceride transfer activity of MTP may be a feasible therapeutic approach for dyslipidemia. 412 42 43 3 lipids to an acceptor membrane. The transfer of lipids occurs down a concentration gradient 76 and does not require energy (Atzel and Wetterau, 1993, 1994). While vertebrate MTP 77 predominantly transfers TG (Rava et al., 2005; Wetterau and Zilversmit, 1985), the Drosophila 78 orthologue of MTP lacks TG transfer activity (Rava et al., 2006), has phospholipid transfer 79 activity and supports secretion of vertebrate ApoB (Khatun et al., 2012; Rava and Hussain, 80 2007; Rava et al., 2006; Sellers et al., 2003). A further analysis of MTTP orthologues in 81 divergent species, including nematodes, insects, fish, and mammals, indicates that all 82 orthologues bind PDI, localize to the ER, and support human ApoB secretion (Rava and 83 Hussain, 2007). However, only vertebrate MTP orthologues exhibit TG transfer activity, 84 suggesting that phospholipid transfer activity was the original function of MTP orthologues and 85 that neutral lipid transfer first evolved in fish (Rava and Hussain, 2007). 87Here we describe a hypomorphic missense mutation in the C-terminal domain of zebrafish mttp 88 (G863V) that decreases the production and size of B-lps in vivo, but has minimal effects on lipid 89 malabsorption in the intestine and no effect on growth. Biochemical characterization of the 90 G863V allele indicates that it is defective in triglyceride transfer activity, but retains phospholipid 91 transfer activity. Further, we show that mutation of the conserved glycine at position 865 in 92 human MTTP also selectively abolishes triglyceride transfer activity. Taken together, these data 93 pr...

Heme is a prosthetic group that plays a critical role in catalyzing life-essential redox reactions in all cells, including critical metabolic processes. Heme synthesis must be tightly co-regulated with cellular requirements in order to maximize utilization and minimize toxicity. Terminally differentiating erythroid cells have an extremely high demand for heme for hemoglobin synthesis. While the enzymatic reactions of heme synthesis are extremely well studied, the mechanisms by which the mitochondrial homeostatic machinery interacts with and regulates heme synthesis are poorly understood. Knowledge of these regulatory mechanisms are key to understanding how red cells couple heme production with heme demand. Heme synthesis is tightly regulated by the mitochondrial AAA+ unfoldase CLPX, which has been reported to promote heme synthesis by activation of yeast δ-aminolevulinate synthase (ALAS/Hem1). CLPX was also reported to mediate heme-induced turnover of ALAS1 in human cells. However, a mutation in the ATP binding domain of CLPX that abrogated ATP binding caused an increase in ALAS activity, contrary to previous predictions that CLPX activated ALAS. Using loss-of-function assays in murine cells and zebrafish, we interrogated the mechanisms by which CLPX regulates erythroid heme synthesis. We found that consistent with previous studies, CLPX is required for erythroid heme synthesis. We show that ALAS2 stability and activity were both increased in the absence of CLPX, suggesting that CLPX primarily regulates ALAS2 by control of its turnover. However, we also showed that CLPX is required for PPOX activity and maintenance of FECH levels, likely accounting for the heme deficiency in the absence of CLPX. Lastly, CLPX is required for iron metabolism during erythroid terminal differentiation. Our results show that the role of CLPX in heme synthesis is not conserved across eukaryotes. Our studies reveal a potential mechanism for the role of CLPX in anemia and porphyria, and reveal multiple nodes at which heme synthesis is regulated by the mitochondrial housekeeping machinery.

Erythroid cells are the main driver of iron utilization in vertebrates, as their main role is to synthesize hemoglobin to oxygenate the body's tissues. As such, iron is a key nutrient for the development and function of erythroid cells. When iron deficient, erythroid cells are both lacking in hemoglobin and exhibit differentiation defects. Currently, the efficacy of iron supplementation is monitored by measuring indices of erythroid hemoglobinization. However, its effect on erythroid differentiation is less clear. In this study, we used zebrafish with genetic iron metabolism defects to determine if iron supplementation could rescue erythropoietic defects in organisms that are iron deficient at the cellular and systemic level. To carry out this study, we developed a technique to sort Tg(globin lcr:EGFP) erythrocytes from single zebrafish embryos onto slides for imaging. We found that iron supplementation in mfrn1 mutant zebrafish, which carry a defect in mitochondrial iron trafficking, restored hemoglobinization but not erythroid cell number or terminal differentiation. Iron supplementation in fpn1 mutant zebrafish, which have defects in export of iron from yolk syncytial cells and intestinal epithelium, functioning a model of dietary iron deficiency, restored erythroid cell number but not terminal differentiation deficiencies. Our data suggests that in addition to adequate iron levels, correct regulation of iron trafficking is required for optimal erythroid iron utilization and terminal erythropoiesis.

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