A vesicular transport pathway shuttles cargo from mitochondria to lysosomes

Mitochondrial respiration relies on electron transport, an essential yet dangerous process in that it leads to the generation of reactive oxygen species (ROS). ROS can be neutralized within the mitochondria through enzymatic activity, yet the mechanism for steady-state removal of oxidized mitochondrial protein complexes and lipids is not well understood. We have previously characterized vesicular profiles budding from the mitochondria that carry selected cargo. At least one population of these mitochondria-derived vesicles (MDVs) targets the peroxisomes; however, the fate of the majority of MDVs was unclear. Here, we demonstrate that MDVs carry selected cargo to the lysosomes. Using a combination of confocal and electron microscopy, we observe MDVs in steady state and demonstrate that they are stimulated as an early response to oxidative stress, the extent of which is determined by the respiratory status of the mitochondria. Delivery to the lysosomes does not require mitochondrial depolarization and is independent of ATG5 and LC3, suggesting that vesicle delivery complements mitophagy. Consistent with this, ultrastructural analysis of MDV formation revealed Tom20-positive structures within the vesicles of multivesicular bodies. These data characterize a novel vesicle transport route between the mitochondria and lysosomes, providing insights into the basic mechanisms of mitochondrial quality control.     

Special delivery from mitochondria to peroxisomes

Inter-organellar communication and interactions are necessary and accepted consequences of the segregation of biochemical functions into subcellular organelles. Recently, Heidi McBride and her collaborators found a novel link between mitochondria and peroxisomes in their discovery of mitochondria-derived vesicles (MDVs), which appear to fuse with a fraction of pre-existing peroxisomes in mammalian cells. We discuss the potential role of this vesicle population in the context of pathways for the exchange of metabolites and/or macromolecules between these compartments.     

Phosphatidylserine transport to the mitochondria is regulated by ubiquitination

Mitochondrial membrane biogenesis requires the interorganelle transport of phospholipids. Phosphatidylserine (PtdSer) synthesized in the endoplasmic reticulum and related membranes (mitochondria-associated membrane (MAM)) is transported to the mitochondria by unknown gene products and decarboxylated to form phosphatidylethanolamine at the inner membrane by PtdSer decarboxylase 1 (Psd1p). We have designed a screen for strains defective in PtdSer transport (pstA mutants) between the endoplasmic reticulum and Psd1p that relies on isolating ethanolamine auxotrophs in suitable (psd2Delta) genetic backgrounds. Following chemical mutagenesis, we isolated an ethanolamine auxotroph that we designate pstA1-1. Using in vivo and in vitro phospholipid synthesis/transport measurements, we demonstrate that the pstA1-1 mutant is defective in PtdSer transport between the MAM and mitochondria. The gene that complements the growth defect and PtdSer transport defect of the pstA1-1 mutant is MET30, which encodes a substrate recognition subunit of the SCF (suppressor of kinetochore protein 1, cullin, F-box) ubiquitin ligase complex. Reconstitution of different permutations of MAM and mitochondria from wild type and pstA1-1 strains demonstrates that the MET30 gene product affects both organelles. These data provide compelling evidence that interorganelle PtdSer traffic is regulated by ubiquitination.     

Targeting of hFis1 to peroxisomes is mediated by Pex19p

The processes of peroxisome formation and proliferation are still a matter of debate. We have previously shown that peroxisomes share some components of their division machinery with mitochondria. hFis1, a tail-anchored membrane protein, regulates the membrane fission of both organelles by DLP1/Drp1 recruitment, but nothing is known about the mechanisms of the dual targeting of hFis1. Here we demonstrate for the first time that peroxisomal targeting of hFis1 depends on Pex19p, a peroxisomal membrane protein import factor. hFis1/Pex19p binding was demonstrated by expression and co-immunoprecipitation studies. Using mutated versions of hFis1 an essential binding region for Pex19p was located within the last 26 C-terminal amino acids of hFis1, which are required for proper targeting to both mitochondria and peroxisomes. The basic amino acids in the very C terminus are not essential for Pex19p binding and peroxisomal targeting, but are instead required for mitochondrial targeting. Silencing of Pex19p by small interference RNA reduced the targeting of hFis1 to peroxisomes, but not to mitochondria. In contrast, overexpression of Pex19p alone was not sufficient to shift the targeting of hFis1 to peroxisomes. Our findings indicate that targeting of hFis1 to peroxisomes and mitochondria are independent events and support a direct, Pex19p-dependent targeting of peroxisomal tail-anchored proteins.     

Bupivacaine myotoxicity is mediated by mitochondria.

We have investigated the effects of the myotoxic local anesthetic bupivacaine on rat skeletal muscle mitochondria and isolated myofibers from flexor digitorum brevis, extensor digitorum longus, soleus, and from the proximal, striated portion of the esophagus. In isolated mitochondria, bupivacaine caused a concentration-dependent mitochondrial depolarization and pyridine nucleotide oxidation, which were matched by an increased oxygen consumption at bupivacaine concentrations of 1.5 mm or less at pH 7.4, whereas respiration was inhibited at higher concentrations. As a consequence of depolarization, bupivacaine caused the opening of the permeability transition pore (PTP), a cyclosporin A-sensitive inner membrane channel that plays a key role in many forms of cell death. In intact flexor digitorum brevis fibers bupivacaine caused mitochondrial depolarization and pyridine nucleotides oxidation that were matched by increased concentrations of cytosolic free Ca(2+), release of cytochrome c, and eventually, hypercontracture. Both mitochondrial depolarization and cytochrome c release were inhibited by cyclosporin A, indicating that PTP opening rather than bupivacaine as such was responsible for these events. Similar responses to bupivacaine were observed in the soleus, which is highly oxidative. In contrast, fibers from the esophagus (which we show to be more fatigable than flexor digitorum brevis fibers) and from the highly glycolytic extensor digitorum longus didn't undergo pyridine nucleotide oxidation upon the addition of bupivacaine and were resistant to bupivacaine toxicity. These results suggest that active oxidative metabolism is a key determinant in bupivacaine toxicity, that bupivacaine myotoxicity is a relevant model of mitochondrial dysfunction involving the PTP and Ca(2+) dysregulation, and that it represents a promising system to test new PTP inhibitors that may prove relevant in spontaneous myopathies where mitochondria have long been suspected to play a role.     

Contribution of vesicular and micellar carriers to cholesterol transport in human bile

A nonmicellar, bile salt-independent mode of cholesterol transport in human bile involving phospholipid vesicles was recently reported by our group. In the present study, we have investigated the relative contribution of the phospholipid vesicles and mixed bile salt-phospholipid micelles to cholesterol transport in human hepatic and gallbladder biles. The vesicles (ca 800 A diameter) were demonstrated by quasi-elastic light scattering (QELS) in fresh bile and after chromatography. Gel filtration under conditions that preserved micellar integrity demonstrated that biliary cholesterol was associated with both vesicles and micelles. At low bile salt concentration, the vesicular phase was predominant and most of the cholesterol was transported by it. With increasing bile salt concentrations, a progressive solubilization of the vesicles occurred with a concomitant increase in the amount of cholesterol transported by micelles. The vesicular carrier may be of particular biological significance for cholesterol solubilization in supersaturated biles.     

Vps10p transport from the trans-Golgi network to the endosome is mediated by clathrin-coated vesicles

A native immunoisolation procedure has been used to investigate the role of clathrin-coated vesicles (CCVs) in the transport of vacuolar proteins between the trans-Golgi network (TGN) and the prevacuolar/endosome compartments in the yeast Saccharomyces cerevisiae. We find that Apl2p, one large subunit of the adaptor protein-1 complex, and Vps10p, the carboxypeptidase Y vacuolar protein receptor, are associated with clathrin molecules. Vps10p packaging in CCVs is reduced in pep12 Delta and vps34 Delta, two mutants that block Vps10p transport from the TGN to the endosome. However, Vps10p sorting is independent of Apl2p. Interestingly, a Vps10C(t) Delta p mutant lacking its C-terminal cytoplasmic domain, the portion of the receptor responsible for carboxypeptidase Y sorting, is also coimmunoprecipitated with clathrin. Our results suggest that CCVs mediate Vps10p transport from the TGN to the endosome independent of direct interactions between Vps10p and clathrin coats. The Vps10p C-terminal domain appears to play a principal role in retrieval of Vps10p from the prevacuolar compartment rather than in sorting from the TGN.     

MEKK1-induced apoptosis is mediated by Smac/Diablo release from the mitochondria

During apoptotic stimulation, the serine threonine kinase, MEKK1, is cleaved into an activated 91 kDa kinase fragment. This cleavage is mediated by caspase 3 and leads to further caspase 3 activation and apoptosis. Forced expression of the 91 kDa kinase fragment induces apoptosis through changes in membrane potential of the mitochondria mediated by permeability transition pore opening. MEKK1 activation, however, fails to release cytochrome c from the mitochondria. Herein, we determined that overexpression of MEKK1 causes mitochondrial Smac/Diablo release correlating with MEKK1-induced apoptosis. Furthermore, using siRNA that lowers Smac/Diablo expression, MEKK1-induced apoptosis was significantly reduced. Mouse embryonic fibroblast cells lacking MEKK1 expression are also resistant to etoposide-induced mitochondrial Smac/Diablo release. In contrast, etoposide-induced mitochondrial cytochrome c release was not inhibited. MEKK1 also activates the MAP kinase JNK, but MEKK1-induced mitochondrial Smac/Diablo release and apoptosis are independent of MEKK1 mediated JNK activation. Taken together, release of Smac/Diablo from the mitochondria plays a role in MEKK1-induced apoptosis.     

Evidence for sustained ATP release from liver cells that is not mediated by vesicular exocytosis

Extracellular ATP regulates many important cellular functions in the liver by stimulating purinergic receptors. Recent studies have shown that rapid exocytosis of ATP-enriched vesicles contributes to ATP release from liver cells. However, this rapid ATP release is transient, and ceases in ~30 s after the exposure to hypotonic solution. The purpose of these studies was to assess the role of vesicular exocytosis in sustained ATP release. An exposure to hypotonic solution evoked sustained ATP release that persisted for more than 15 min after the exposure. Using FM1-43 (N-(3-triethylammoniumpropyl)-4-(4-(dibutylamino)styryl)pyridinium dibromide) fluorescence to measure exocytosis, we found that hypotonic solution stimulated a transient increase in FM1-43 fluorescence that lasted ~2 min. Notably, the rate of FM1-43 fluorescence and the magnitude of ATP release were not correlated, indicating that vesicular exocytosis may not mediate sustained ATP release from liver cells. Interestingly, mefloquine potently inhibited sustained ATP release, but did not inhibit an increase in FM1-43 fluorescence evoked by hypotonic solution. Consistent with these findings, when exocytosis of ATP-enriched vesicles was specifically stimulated by 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB), mefloquine failed to inhibit ATP release evoked by NPPB. Thus, mefloquine can pharmacologically dissociate sustained ATP release and vesicular exocytosis. These results suggest that a distinct mefloquine-sensitive membrane ATP transport may contribute to sustained ATP release from liver cells. This novel mechanism of membrane ATP transport may play an important role in the regulation of purinergic signaling in liver cells.     

ER-to-Golgi transport: form and formation of vesicular and tubular carriers

The transport of proteins and lipids between the endoplasmic reticulum and Golgi apparatus is initiated by the collection of secretory cargo from within the lumen of the endoplasmic reticulum. Subsequently, transport carriers are formed that bud from this membrane and are transported to, and subsequently merge with, the Golgi. The principle driving force behind the budding process is the multi-subunit coat protein complex, COPII. A considerable amount of information is now available regarding the molecular mechanisms by which COPII components operate together to drive cargo selection and transport carrier formation. In contrast, the precise nature of the transport carriers formed is still a matter of considerable debate. Vesicular and tubular carriers have been characterized that are, or in other cases are not, coated with the COPII complex. Here, we seek to integrate much of the data surrounding this topic and try to understand the mechanisms by which vesicular and/or tubular carriers might be generated.     

Taking vesicular transport to the cilium

Defects in protein trafficking within the cell body and cilia are thought to underlie the human disease Bardet-Biedl syndrome (BBS). In this issue, Nachury et al. (2007) reveal that a large complex of proteins implicated in BBS cooperates with Rabin8-the GTP exchange factor for the small GTPase Rab8-to promote cilia formation and presumably movement of membrane proteins from the cell into the cilium.     

Porphyrin accumulation in mitochondria is mediated by 2-oxoglutarate carrier

Heme (Fe-protoporphyrin IX), an endogenous porphyrin derivative, is an essential molecule in living aerobic organisms and plays a role in a variety of physiological processes such as oxygen transport, respiration, and signal transduction. For the biosynthesis of heme or the mitochondrial heme proteins, heme or its biosynthetic precursor porphyrin must be transported into mitochondria from cytosol. The mechanism of porphyrin accumulation in the mitochondrial inner membrane is unclear. In the present study, we analyzed the mechanism of mitochondrial translocation of porphyrin derivatives. We showed that palladium meso-tetra(4-carboxyphenyl)porphyrin (PdTCPP), a phosphorescent porphyrin derivative, accumulated in the mitochondria of several cell lines. Using affinity latex beads, we showed that 2-oxoglutarate carrier (OGC), the mitochondrial transporter of 2-oxoglutarate, bound to PdTCPP, and in vitro PdTCPP inhibited 2-oxoglutarate uptake into mitochondria in a competitive manner (Ki = 15 microM). Interestingly, all types of porphyrin derivatives examined in this study competitively inhibited 2-oxoglutarate uptake into mitochondria, including protoporphyrin IX, coproporphyrin III, and hemin. Furthermore, mitochondrial accumulation of porphyrins was inhibited by 2-oxoglutarate or OGC inhibitor. These results suggested that porphyrin accumulation in mitochondria is mediated by OGC and that porphyrins are able to competitively inhibit 2-oxoglutarate uptake into mitochondria. This is the first report of a putative mechanism for accumulation of porphyrins in the mitochondrial inner membrane.     

Biogenesis of peroxisomes and mitochondria: linked by division

Peroxisomes and mitochondria are metabolically linked organelles, which are crucial to human health and development. The search for components involved in their dynamics and maintenance led to the interesting finding that mitochondria and peroxisomes share components of their division machinery. Recently, it became clear that this is a common strategy used by mammals, fungi and plants. Furthermore, a closer interrelationship between peroxisomes and mitochondria has been proposed, which might have an impact on functionality and disease conditions. Here, we briefly highlight the major findings, views and open questions concerning peroxisomal formation, division, and interrelationship with mitochondria. Presented at the 50th Anniversary Symposium of the Society for Histochemistry, Interlaken, Switzerland, October 1–4, 2008.     

Magnesium transport by mitochondria

The pathways for the uptake and extrusion of Mg²⁺ by mitochondria are not well defined. the present evidence suggests that uptake occurs by nonspecific diffusive pathways in response to elevated membrane potential. There is disagreement as to some of the properties of Mg²⁺ efflux from mitochondria, but the reaction resembles K⁺ efflux in many ways and may occur in exchange for H⁺. Matrix free magnesium ion concentration, [Mg²⁺], can be measured using fluorescent probes and is set very close to cytosol [Mg²⁺] by a balance between influx and efflux and by the availability of ligands, such as P_i. There are indications that matrix [Mg²⁺] may be under hormonal control and that it contributes to the regulation of mitochondrial metabolism and transport reactions.     

VAMP-7 mediates vesicular transport from endosomes to lysosomes.

A more complete picture of the molecules that are critical for the organization of membrane compartments is beginning to emerge through the characterization of proteins in the vesicle-associated membrane protein (also called synaptobrevin) family of membrane trafficking proteins. To better understand the mechanisms of membrane trafficking within the endocytic pathway, we generated a series of monoclonal and polyclonal antibodies against the cytoplasmic domain of vesicle-associated membrane protein 7 (VAMP-7). The antibodies recognize a 25-kD membrane-associated protein in multiple tissues and cell lines. Immunohistochemical analysis reveals colocalization with a marker of late endosomes and lysosomes, lysosome-associated membrane protein 1 (LAMP-1), but not with other membrane markers, including p115 and transferrin receptor. Treatment with nocodozole or brefeldin A does not disrupt the colocalization of VAMP-7 and LAMP-1. Immunoelectron microscopy analysis shows that VAMP-7 is most concentrated in the trans-Golgi network region of the cell as well as late endosomes and transport vesicles that do not contain the mannose-6 phosphate receptor. In streptolysin- O-permeabilized cells, antibodies against VAMP-7 inhibit the breakdown of epidermal growth factor but not the recycling of transferrin. These data are consistent with a role for VAMP-7 in the vesicular transport of proteins from the early endosome to the lysosome.     

Multiple GTP-binding proteins regulate vesicular transport from the ER to Golgi membranes

Using indirect immunofluorescence we have examined the effects of reagents which inhibit the function of ras-related rab small GTP-binding proteins and heterotrimeric G alpha beta gamma proteins in ER to Golgi transport. Export from the ER was inhibited by an antibody towards rab1B and an NH2-terminal peptide which inhibits ARF function (Balch, W. E., R. A. Kahn, and R. Schwaninger. 1992. J. Biol. Chem. 267:13053-13061), suggesting that both of these small GTP-binding proteins are essential for the transport vesicle formation. Export from the ER was also potently inhibited by mastoparan, a peptide which mimics G protein binding regions of seven transmembrane spanning receptors activating and uncoupling heterotrimeric G proteins from their cognate receptors. Consistent with this result, purified beta gamma subunits inhibited the export of VSV-G from the ER suggesting an initial event in transport vesicle assembly was regulated by a heterotrimeric G protein. In contrast, incubation in the presence of GTP gamma S or AIF(3-5) resulted in the accumulation of transported protein in different populations of punctate pre-Golgi intermediates distributed throughout the cytoplasm of the cell. Finally, a peptide which is believed to antagonize the interaction of rab proteins with putative downstream effector molecules inhibited transport at a later step preceding delivery to the cis Golgi compartment, similar to the site of accumulation of transported protein in the absence of NSF or calcium (Plutner, H., H. W. Davidson, J. Saraste, and W. E. Balch. 1992. J. Cell Biol. 119:1097-1116). These results are consistent with the hypothesis that multiple GTP-binding proteins including a heterotrimeric G protein(s), ARF and rab1 differentially regulate steps in the transport of protein between early compartments of the secretory pathway. The concept that G protein-coupled receptors gate the export of protein from the ER is discussed.     

Nuclear import of Ran is mediated by the transport factor NTF2

A concentration gradient of the GTP-bound form of the GTPase Ran across nuclear pores is essential for the transport of many proteins and nucleic acids between the nuclear and cytoplasmic compartments of eukaryotic cells [1], [2], [3] and [4]. The mechanisms responsible for the dynamics and maintenance of this Ran gradient have been unclear. We now show that Ran shuttles between the nucleosol and cytosol, and that cytosolic Ran accumulates rapidly in the nucleus in a saturable manner that is dependent on temperature and on the guanine-nucleotide exchange factor RCC1. Nuclear import in digitonin-permeabilized cells in the absence of added factors was minimal. The addition of energy and nuclear transport factor 2 (NTF2) [5] was sufficient for the accumulation of Ran in the nucleus. An NTF2 mutant that cannot bind Ran [6] was unable to facilitate Ran import. A GTP-bound form of a Ran mutant that cannot bind NTF2 was not a substrate for import. A dominant-negative importin-β mutant inhibited nuclear import of Ran, whereas addition of transportin, which accumulates in the nucleus, enhanced NTF2-dependent Ran import. We conclude that NTF2 functions as a transport receptor for Ran, permitting rapid entry into the nucleus where GTP-GDP exchange mediated by RCC1 [7] converts Ran into its GTP-bound state. The Ran–GTP can associate with nuclear Ran-binding proteins, thereby creating a Ran gradient across nuclear pores.     

Cytoplasmic fungal lipases release fungicides from ultra-deformable vesicular drug carriers

The Transfersome? is a lipid vesicle that contains membrane softeners, such as Tween 80, to make it ultra-deformable. This feature makes the Transfersome? an efficient carrier for delivery of therapeutic drugs across the skin barrier. It was reported that TDT 067 (a topical formulation of 15 mg/ml terbinafine in Transfersome? vesicles) has a much more potent antifungal activity in vitro compared with conventional terbinafine, which is a water-insoluble fungicide. Here we use ultra-structural studies and live imaging in a model fungus to describe the underlying mode of action. We show that terbinafine causes local collapse of the fungal endoplasmic reticulum, which was more efficient when terbinafine was delivered in Transfersome? vesicles (TFVs). When applied in liquid culture, fluorescently labeled TFVs rapidly entered the fungal cells (T(1/2)~2 min). Entry was F-actin- and ATP-independent, indicating that it is a passive process. Ultra-structural studies showed that passage through the cell wall involves significant deformation of the vesicles, and depends on a high concentration of the surfactant Tween 80 in their membrane. Surprisingly, the TFVs collapsed into lipid droplets after entry into the cell and the terbinafine was released from their interior. With time, the lipid bodies were metabolized in an ATP-dependent fashion, suggesting that cytosolic lipases attack and degrade intruding TFVs. Indeed, the specific monoacylglycerol lipase inhibitor URB602 prevented Transfersome? degradation and neutralized the cytotoxic effect of Transfersome?-delivered terbinafine. These data suggest that (a) Transfersomes deliver the lipophilic fungicide Terbinafine to the fungal cell wall, (b) the membrane softener Tween 80 allows the passage of the Transfersomes into the fungal cell, and (c) fungal lipases digest the invading Transfersome? vesicles thereby releasing their cytotoxic content. As this mode of action of Transfersomes is independent of the drug cargo, these results demonstrate the potential of Transfersomes in the treatment of all fungal diseases.