The intestinal cells of Caenorhabditis elegans embryos contain prominent, birefringent gut granules that we show are lysosome-related organelles. Gut granules are labeled by lysosomal markers, and their formation is disrupted in embryos depleted of AP-3 subunits, VPS-16, and VPS-41. We define a class of gut granule loss (glo) mutants that are defective in gut granule biogenesis. We show that the glo-1 gene encodes a predicted Rab GTPase that localizes to lysosome-related gut granules in the intestine and that glo-4 encodes a possible GLO-1 guanine nucleotide exchange factor. These and other glo genes are homologous to genes implicated in the biogenesis of specialized, lysosome-related organelles such as melanosomes in mammals and pigment granules in Drosophila. The glo mutants thus provide a simple model system for the analysis of lysosome-related organelle biogenesis in animal cells. INTRODUCTIONLysosomes are ubiquitous membrane-bound organelles that function as major degradative sites within eukaryotic cells (Tappel, 1969). Lysosomes contain an assortment of aciddependent hydrolases that function in the breakdown of proteins, lipids, nucleic acids, and oligosaccharides. Lysosomes receive exogenous material through the endocytic pathway and are characterized as being the terminal compartment of the endocytic pathway. Lysosomes also receive material via the secretory pathway and directly from the cytoplasm (Kornfeld and Mellman, 1989;Mullins and Bonifacino, 2001;Luzio et al., 2003). Lysosomes function in diverse and important cellular processes including cell surface receptor turnover, destruction of pathogens, antigen processing, digestion, starvation responses, tissue remodeling, ion storage, autophagy, and plasma membrane repair.The yeast vacuole shares several characteristics with the lysosomes of higher animals. Genetic screens have led to the identification of Ͼ150 genes necessary for the transport and sorting of newly synthesized proteins to the yeast vacuole (Jones, 1977;Bankaitis et al., 1986;Rothman and Stevens, 1986;Bonangelino et al., 2002). These genes control two pathways of Golgi-to-vacuole transport, the carboxypeptidase Y (CPY) and alkaline phosphatase (ALP) sorting pathways (Burd et al., 1998;Conibear and Stevens, 1998;Mullins and Bonifacino, 2001). Proteins trafficked via the CPY pathway transit an endosomal prevacuolar compartment en route to the vacuole. The ALP pathway mediates transport to the vacuole independent of the prevacuolar compartment.Many of the genes involved in transport to the yeast vacuole have homologues in higher animals (Lemmon and Traub, 2000;Mullins and Bonifacino, 2001;Bonangelino et al., 2002). For example, the HOPS complex proteins (Vps11p, Vps16p, Vps18p, and Vps33p) regulate membrane fusion events necessary for lysosomal delivery within yeast (Rieder and Emr, 1997;Peterson and Emr, 2001), Drosophila melanogaster (Sevrioukov et al., 1999;Sriram et al., 2003), and mammalian (Poupon et al., 2003;Richardson et al., 2004) endosomal systems. Similarly, the proteins compos...
Membrane fusion is required to establish the morphology and cellular distribution of the mitochondrial compartment. In Drosophila, mutations in the fuzzy onions (fzo) GTPase block a developmentally regulated mitochondrial fusion event during spermatogenesis. Here we report that the yeast orthologue of fuzzy onions, Fzo1p, plays a direct and conserved role in mitochondrial fusion. A conditional fzo1 mutation causes the mitochondrial reticulum to fragment and blocks mitochondrial fusion during yeast mating. Fzo1p is a mitochondrial integral membrane protein with its GTPase domain exposed to the cytoplasm. Point mutations that alter conserved residues in the GTPase domain do not affect Fzo1p localization but disrupt mitochondrial fusion. Suborganellar fractionation suggests that Fzo1p spans the outer and is tightly associated with the inner mitochondrial membrane. This topology may be required to coordinate the behavior of the two mitochondrial membranes during the fusion reaction. We propose that the fuzzy onions family of transmembrane GTPases act as molecular switches to regulate a key step in mitochondrial membrane docking and/or fusion.
The Saccharomyces cerevisiae Dnm1 protein is structurally related to dynamin, a GTPase required for membrane scission during endocytosis. Here we show that Dnm1p is essential for the maintenance of mitochondrial morphology. Disruption of the DNM1 gene causes the wild-type network of tubular mitochondrial membranes to collapse to one side of the cell but does not affect the morphology or distribution of other cytoplasmic organelles. Dnm1 proteins containing point mutations in the predicted GTP-binding domain or completely lacking the GTP-binding domain fail to rescue mitochondrial morphology defects in a dnm1 mutant and induce dominant mitochondrial morphology defects in wild-type cells. Indirect immunofluorescence reveals that Dnm1p is distributed in punctate structures at the cell cortex that colocalize with the mitochondrial compartment. These Dnm1p-containing structures remain associated with the spherical mitochondria found in an mdm10 mutant strain. In addition, a portion of Dnm1p cofractionates with mitochondrial membranes during differential sedimentation and sucrose gradient fractionation of wild-type cells. Our results demonstrate that Dnm1p is required for the cortical distribution of the mitochondrial network in yeast, a novel function for a dynamin-related protein.
The intestine of Caenorhabditis elegans is an epithelial tube consisting of only 20 cells and is derived clonally from a single embryonic blastomere called E. We describe the cellular events that shape the intestine. These events include cytoplasmic polarization of cells in the intestinal primordium, the intercalation of specific sets of cells, the generation of an extracellular cavity within the primordium, and adherens junction formation. The polarization of the intestinal primordium is associated with the generation of an asymmetric microtubule cytoskeleton, and microtubule function plays a role in subsequent cell polarity. We show that an isolated E blastomere is capable of generating polarized intestinal cells, indicating that some of the major events in intestinal organogenesis do not depend upon interactions with surrounding tissues. We compare and contrast intestinal organogenesis with some of the basic steps in development of a second epithelial organ, the pharynx, and suggest how these differences lead to organs with distinct shapes.
Caenorhabditis elegans gut granules are intestine specific lysosome-related organelles with birefringent and autofluorescent contents. We identified pgp-2, which encodes an ABC transporter, in screens for genes required for the proper formation of gut granules. pgp-2(-) embryos mislocalize birefringent material into the intestinal lumen and are lacking in acidified intestinal V-ATPase-containing compartments. Adults without pgp-2(+) function similarly lack organelles with gut granule characteristics. These cellular phenotypes indicate that pgp-2(-) animals are defective in gut granule biogenesis. Double mutant analysis suggests that pgp-2(+) functions in parallel with the AP-3 adaptor complex during gut granule formation. We find that pgp-2 is expressed in the intestine where it functions in gut granule biogenesis and that PGP-2 localizes to the gut granule membrane. These results support a direct role of an ABC transporter in regulating lysosome biogenesis. Previously, pgp-2(+) activity has been shown to be necessary for the accumulation of Nile Red-stained fat in C. elegans. We show that gut granules are sites of fat storage in C. elegans embryos and adults. Notably, levels of triacylglycerides are relatively normal in animals defective in the formation of gut granules. Our results provide an explanation for the loss of Nile Red-stained fat in pgp-2(-) animals as well as insight into the specialized function of this lysosome-related organelle.
In Saccharomyces cerevisiae, the growing bud inherits a portion of the mitochondrial network from the mother cell soon after it emerges. Although this polarized transport of mitochondria is thought to require functions of the cytoskeleton, there are conflicting reports concerning the nature of the cytoskeletal element involved. Here we report the isolation of a yeast mutant, mdm20, in which both mitochondrial inheritance and actin cables (bundles of actin filaments) are disrupted. The MDM20 gene encodes a 93-kD polypeptide with no homology to other characterized proteins. Extra copies of TPM1, a gene encoding the actin filament–binding protein tropomyosin, suppress mitochondrial inheritance defects and partially restore actin cables in mdm20Δ cells. Synthetic lethality is also observed between mdm20 and tpm1 mutant strains. Overexpression of a second yeast tropomyosin, Tpm2p, rescues mutant phenotypes in the mdm20 strain to a lesser extent. Together, these results provide compelling evidence that mitochondrial inheritance in yeast is an actin-mediated process. MDM20 and TPM1 also exhibit the same pattern of genetic interactions; mutations in MDM20 are synthetically lethal with mutations in BEM2 and MYO2 but not SAC6. Although MDM20 and TPM1 are both required for the formation and/or stabilization of actin cables, mutations in these genes disrupt mitochondrial inheritance and nuclear segregation to different extents. Thus, Mdm20p and Tpm1p may act in vivo to establish molecular and functional heterogeneity of the actin cytoskeleton.
Proteins that control mitochondrial dynamics in yeast are being identified at a rapid pace. These proteins include cytoskeletal elements that regulate organelle distribution and inheritance and several outer membrane proteins that are required to maintain the branched, mitochondrial reticulum. Interestingly, three of the high molecular weight GTPases encoded by the yeast genome are required for mitochondrial integrity and are potential regulators of mitochondrial branching, distribution, and membrane fusion. The recent finding that mtDNA mixing is restricted in the mitochondrial matrix has stimulated the hunt for the molecular machinery that anchors mitochondrial nucleoids in the organelle. Considering that many aspects of mitochondrial structure and behavior are strikingly similar in different cell types, the functional analyses of these yeast proteins should provide general insights into the mechanisms governing mitochondrial dynamics in all eukaryotes.
This work presents a systematic analysis of how VPS-C/HOPS, CCZ-1/SAND-1, and RAB-7, which have well-defined roles in lysosome formation, act in the biogenesis of Caenorhabditis elegans lysosome-related organelles. It identifies key molecular similarities and differences in trafficking to these homologous, yet distinct organelles.
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