The exon 8-containing prosaposin gene splice variant is dispensable for mouse development, lysosomal function, and secretion.

Prosaposin is a multifunctional protein with diverse functions. Intracellularly, prosaposin is a precursor of four sphingolipid activator proteins, saposins A to D, which are required for hydrolysis of sphingolipids by several lysosomal exohydrolases. Secreted prosaposin has been implicated as a neurotrophic, myelinotrophic, and myotrophic factor as well as a spermatogenic factor. It has also been implicated in fertilization. The human and the mouse prosaposin gene has a 9-bp exon (exon 8) that is alternatively spliced, resulting in an isoform with three extra amino acids, Gln-Asp-Gln, within the saposin B domain. Alternative splicing in the prosaposin gene is conserved from fish to humans, tissue specific, and regulated in the brain during development and nerve regeneration-degeneration processes. To elucidate the physiological role of alternative splicing, we have generated a mouse lacking exon 8 by homologous recombination. The exon 8 prosaposin mutant mice are healthy and fertile with no obvious phenotype. No changes were detected in prosaposin secretion or in accumulation and metabolism of gangliosides, sulfatides, neutral glycosphingolipids, neutral phospholipids, other neutral lipids, and ceramide. These data strongly indicate that the prosaposin variant containing the exon 8-encoded three amino acids is dispensable for normal mouse development and fertility as well as for prosaposin secretion and its lysosomal function, at least in the presence of the prosaposin variant missing the exon 8-encoded three amino acids.

EHDS are serine phosphoproteins: EHD1 phosphorylation is enhanced by serum stimulation.

Endocytic processes are mediated by multiple protein-protein interacting modules and regulated by phosphorylation and dephosphorylation. The Eps15 homology domain containing protein 1 (EHD1) has been implicated in regulating recycling of proteins, internalized both in clathrin-dependent and clathrin-independent endocytic pathways, from the recycling compartment to the plasma membrane. EHD1 was found in a complex with clathrin, adaptor protein complex-2 (AP-2) and insulin-like growth factor-1 receptor (IGF-1R), and was shown to interact with Rabenosyn-5, SNAP29, EHBP1 (EH domain binding protein 1) and syndapin I and II. In this study, we show that EHD1, like the other human EHDs, undergoes serine-phosphorylation. Our results also indicate that EHD1 is a serum-inducible serine-phosphoprotein and that PKC (protein kinase C) is one of its kinases. In addition, we show that inhibitors of clathrin-mediated endocytosis decrease EHD1 phosphorylation, while inhibitors of caveolinmediated endocytosis do not affect EHD1 phosphorylation. The results of experiments in which inhibitors of endocytosis were employed strongly suggest that EHD1 phosphorylation occurs between early endosomes and the endocytic recycling compartment.

A Krebs Cycle Component Limits Caspase Activation Rate through Mitochondrial Surface Restriction of CRL Activation.

How cells avoid excessive caspase activity and unwanted cell death during apoptotic caspase-mediated removal of large cellular structures is poorly understood. We investigate caspase-mediated extrusion of spermatid cytoplasmic contents in Drosophila during spermatid individualization. We show that a Krebs cycle component, the ATP-specific form of the succinyl-CoA synthetase ß subunit (A-Sß), binds to and activates the Cullin-3-based ubiquitin ligase (CRL3) complex required for caspase activation in spermatids. In vitro and in vivo evidence suggests that this interaction occurs on the mitochondrial surface, thereby limiting the source of CRL3 complex activation to the vicinity of this organelle and reducing the potential rate of caspase activation by at least 60%. Domain swapping between A-Sß and the GTP-specific SCSß (G-Sß), which functions redundantly in the Krebs cycle, show that the metabolic and structural roles of A-Sß in spermatids can be uncoupled, highlighting a moonlighting function of this Krebs cycle component in CRL activation.

Conservation of expression and alternative splicing in the prosaposin gene.

Prosaposin is the precursor of four lysosomal activator molecules known as saposins A, B, C and D. It is also secreted and was proposed to be a neurotrophic factor. The neurotrophic function was attributed to the amino terminus of saposin C. In man, mouse and rat prosaposin is transcribed to two major isoforms differing in the inclusion of 9 bps of exon 8 within the saposin B domain. In the present study, we show that there is evolutionary conservation of the prosaposin structure and alternative splicing in chick and zebrafish as well. Moreover, there is conservation in prosaposin expression as tested immunohistochemically in the mouse and chick developing brain. We developed a sensitive assay to quantitate the prosaposin alternatively spliced forms. Our results indicate that, in mouse brain, skeletal and cardiac muscle the exon 8-containing RNA is most abundant, while it is almost absent from visceral and smooth muscle-containing organs. We observed temporal and differential expression of the alternatively spliced prosaposin mRNAs in mouse and chick brain as well as during development. The elevation in the abundance of exon 8-containing prosaposin RNA during mouse and chick brain development may suggest a role for the exon 8-containing prosaposin form in this process.

Paternal mitochondrial destruction after fertilization is mediated by a common endocytic and autophagic pathway in Drosophila.

Almost all animals contain mitochondria of maternal origin only, but the exact mechanisms underlying this phenomenon are still vague. We investigated the fate of Drosophila paternal mitochondria after fertilization. We demonstrate that the sperm mitochondrial derivative (MD) is rapidly eliminated in a stereotypical process dubbed paternal mitochondrial destruction (PMD). PMD is initiated by a network of vesicles resembling multivesicular bodies and displaying common features of the endocytic and autophagic pathways. These vesicles associate with the sperm tail and mediate the disintegration of its plasma membrane. Subsequently, the MD separates from the axoneme and breaks into smaller fragments, which are then sequestered by autophagosomes for degradation in lysosomes. We further provide evidence for the involvement of the ubiquitin pathway and the autophagy receptor p62 in this process. Finally, we show that the ubiquitin ligase Parkin is not involved in PMD, implying a divergence from the autophagic pathway of damaged mitochondria.

There is more to life than death: a moonlighting function of a Bcl-2 member.

Members of the Bcl-2 family proteins are best known for their roles in apoptosis regulation. In this issue of Developmental Cell, Popgeorgiev et al. (2011) have uncovered a new, nonapoptotic role for a Bcl-2 homolog during early embryogenesis in zebrafish.