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The transport and intracellular trafficking of heme biosynthesis intermediates are crucial for hemoglobin production, which is a critical process in developing red cells. Here, we profiled gene expression in terminally differentiating murine fetal liver-derived erythroid cells to identify regulators of heme metabolism. We determined that TMEM14C, an inner mitochondrial membrane protein that is enriched in vertebrate hematopoietic tissues, is essential for erythropoiesis and heme synthesis in vivo and in cultured erythroid cells. In mice, TMEM14C deficiency resulted in porphyrin accumulation in the fetal liver, erythroid maturation arrest, and embryonic lethality due to profound anemia. Protoporphyrin IX synthesis in TMEM14C-deficient erythroid cells was blocked, leading to an accumulation of porphyrin precursors. The heme synthesis defect in TMEM14C-deficient cells was ameliorated with a protoporphyrin IX analog, indicating that TMEM14C primarily functions in the terminal steps of the heme synthesis pathway. Together, our data demonstrate that TMEM14C facilitates the import of protoporphyrinogen IX into the mitochondrial matrix for heme synthesis and subsequent hemoglobin production. Furthermore, the identification of TMEM14C as a protoporphyrinogen IX importer provides a genetic tool for further exploring erythropoiesis and congenital anemias.

The spectrin membrane skeleton controls the disposition of selected membrane channels, receptors, and transporters. In the brain [beta]III spectrin binds directly to the excitatory amino acid transporter (EAAT4), the glutamate receptor delta, and other proteins. Mutations in [beta]III spectrin link strongly to human spinocerebellar ataxia type 5 (SCA5), correlating with alterations in EAAT4. We have explored the mechanistic basis of this phenotype by targeted gene disruption of Spnb3. Mice lacking intact [beta]III spectrin develop normally. By 6 months they display a mild nonprogressive ataxia. By 1 year most [Spnb3.sup.-/-] animals develop a myoclonic seizure disorder with significant reductions of EAAT4, EAAT1, GluR[delta], IP3R, and NCAM140. Other synaptic proteins are normal. The cerebellum displays increased dark Purkinje cells (PC), a thin molecular layer, fewer synapses, a loss of dendritic spines, and a 2-fold expansion of the PC dendrite diameter. Membrane and expanded Golgi profiles fill the PC dendrite and soma, and both regions accumulate EAAT4. Correlating with the seizure disorder are enhanced hippocampal levels of neuropeptide Y and EAAT3 and increased calpain proteolysis of [alpha]II spectrin. It appears that [beta]III spectrin disruption impairs synaptogenesis by disturbing the intracellular pathways selectively regulating protein trafficking to the synapse. The mislocalization of these proteins secondarily disrupts glutamate transport dynamics, leading to seizures, neuronal damage, and compensatory changes in EAAT3 and neuropeptide Y. cytoskeleton | membrane | spinocerebellar ataxia type 5 | excitatory amino acid transporter 4 | Purkinje doi/10.1073/pnas.1001522107

Adducins are a family of cytoskeleton proteins encoded by three genes ([Alpha], [Beta], [Gamma]). In a comprehensive assay of gene expression, we show the ubiquitous expression of [Alpha]- and [Gamma]-adducins in contrast to the restricted expression of [Beta]-adducin, [Beta]-adducin is expressed at high levels in brain and hematopoietic tissues (bone marrow in humans, spleen in mice). To elucidate adducin's role in vivo, we created [Beta]-adducin null mice by gene targeting, deleting exons 9-13. A 55-kDa chimeric polypeptide is produced from the first eight exons of [Beta]-adducin and part of the neo cassette in spleen but is not detected in peripheral RBCs or brain, [Beta]-adducin null RBCs are osmotically fragile, spherocytic, and dehydrated compared with the wild type, resembling RBCs from patients with hereditary spherocytosis. The lack of [Beta]-adducin in RBCs leads to decreased membrane incorporation of [Alpha]-adducin (30% of normal) and unexpectedly promotes a 5-fold increase in [Gamma]-adducin incorporation into the RBC membrane skeleton. This study demonstrates adducin's importance to RBC membrane stability in vivo.

The function of the red blood cell anion exchanger, AE1, in in vivo RBC development is investigated by gene targeting. Specifically, null mutation in mice is carried out by targeted gene disruption in embryonic stem cells. The hematological effects of AE1 deletion do not affect fetal loss of homozygotes. Instances of survival to adulthood and fertility are even reported. Results suggest that AE1 is vital to membrane lipid stability but its plasma membrane localization is not required for either terminal RBC differentiation or membrane skeleton assembly.

Lysosomal storage in the spleen and liver, associated with mucopolysaccharidosis (Sly syndrome), can be mitigated by gene therapy in mice. A beta-glucuronidase gene is transferred into mutant hematopoietic stem cells through the mediation of a retroviral vector. This results in the low-level, long-term expression of beta-glucuronidase, reducing lysosomal accumulation in the spleen and liver and increasing the life span of the treated mice.