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1.
Toxoplasma gondii replicates within a specialized vacuole surrounded by the parasitophorous vacuole membrane (PVM). The PVM forms intimate interactions with host mitochondria and endoplasmic reticulum (ER) in a process termed PVM-organelle association. In this study we identify a likely mediator of this process, the parasite protein ROP2. ROP2, which is localized to the PVM, is secreted from anterior organelles termed rhoptries during parasite invasion into host cells. The NH(2)-terminal domain of ROP2 (ROP2hc) within the PVM is exposed to the host cell cytosol, and has characteristics of a mitochondrial targeting signal. In in vitro assays, ROP2hc is partially translocated into the mitochondrial outer membrane and behaves like an integral membrane protein. Although ROP2hc does not translocate across the ER membrane, it does exhibit carbonate-resistant binding to this organelle. In vivo, ROP2hc expressed as a soluble fragment in the cytosol of uninfected cells associates with both mitochondria and ER. The 30-amino acid (aa) NH(2)-terminal sequence of ROP2hc, when fused to green fluorescent protein (GFP), is sufficient for mitochondrial targeting. Deletion of the 30-aa NH(2)-terminal signal from ROP2hc results in robust localization of the truncated protein to the ER. These results demonstrate a new mechanism for tight association of different membrane-bound organelles within the cell cytoplasm.  相似文献   

2.
Characteristics of brefeldin A (BFA)-induced redistribution of Golgi proteins into the endoplasmic reticulum (ER) and its relationship to an ER retrieval pathway were investigated. Retrograde movement of Golgi proteins into the ER occurred via long, tubulovesicular processes extending out of the Golgi along microtubules. Microtubule-disrupting agents (i.e., nocodazole), energy poisons, and reduced temperatures inhibited this pathway. In BFA-treated cells Golgi proteins appeared to cycle between the ER and an intermediate compartment marked by a 53 kd protein. Addition of nocodazole disrupted this dynamic cycle by preferentially inhibiting retrograde movement, causing Golgi proteins to accumulate in the intermediate compartment. In the absence of BFA, such an ER cycling pathway appeared to be followed normally by the 53 kd protein but not by Golgi proteins, as revealed by temperature shift experiments. We propose that BFA induces the interaction of the Golgi with an intermediate "recycling" compartment that utilizes a microtubule-dependent pathway into the ER.  相似文献   

3.
Diacylglycerol is necessary for trans-Golgi network (TGN) to cell surface transport, but its functional relevance in the early secretory pathway is unclear. Although depletion of diacylglycerol did not affect ER-to-Golgi transport, it led to a redistribution of the KDEL receptor to the Golgi, indicating that Golgi-to-ER transport was perturbed. Electron microscopy revealed an accumulation of COPI-coated membrane profiles close to the Golgi cisternae. Electron tomography showed that the majority of these membrane profiles originate from coated buds, indicating a block in membrane fission. Under these conditions the Golgi-associated pool of ARFGAP1 was reduced, but there was no effect on the binding of coatomer or the membrane fission protein CtBP3/BARS to the Golgi. The addition of 1,2-dioctanoyl-sn-glycerol or the diacylglycerol analogue phorbol 12,13-dibutyrate reversed the effects of endogenous diacylglycerol depletion. Our findings implicate diacylglycerol in the retrograde transport of proteins from Golgi to the ER and suggest that it plays a critical role at a late stage of COPI vesicle formation.  相似文献   

4.
《Autophagy》2013,9(1):29-46
When Saccharomyces cerevisiae is starved of glucose, the gluconeogenic enzymes fructose-1,6-bisphosphatase (FBPase), malate dehydrogenase (MDH2), isocitrate lyase (Icl1) and phosphoenolpyruvate carboxykinase (Pck1) are induced. However, when glucose is added to prolonged starved cells, these enzymes are degraded in the vacuole via the vacuole import and degradation (Vid) pathway. Recent evidence suggests that the Vid pathway merges with the endocytic pathway at actin patches where endocytic vesicles are formed. The convergence of the Vid pathway with the endocytic pathway allows cells to remove intracellular and extracellular proteins simultaneously. However, the genes that regulate this step of the convergence have not been identified previously. Here we show that VID30 plays a critical role for the association of Vid vesicles and actin patches. Vid30 is constitutively expressed and interacts with Vid vesicle proteins Vid24 and Sec28 but not with the cargo protein FBPase. In the absence of SEC28 or VID24, Vid30 association with actin patches was prolonged. In cells lacking the VID30 gene, FBPase and Vid24 were not localized to actin patches, suggesting that Vid30 has a role in the association of Vid vesicles and actin patches. Vid30 contains a LisH and a CTLH domain, both of which are required for FBPase degradation. When these domains were deleted, FBPase trafficking to the vacuole was impaired. We suggest that Vid30 also has a role in the Vid pathway at a later step in a process that is mediated by the LisH and CTLH domains.  相似文献   

5.
When Saccharomyces cerevisiae is starved of glucose, the gluconeogenic enzymes fructose-1,6-bisphosphatase (FBPase), malate dehydrogenase (MDH2), isocitrate lyase (Icl1) and phosphoenolpyruvate carboxykinase (Pck1) are induced. However, when glucose is added to prolonged starved cells, these enzymes are degraded in the vacuole via the vacuole import and degradation (Vid) pathway. Recent evidence suggests that the Vid pathway merges with the endocytic pathway at actin patches where endocytic vesicles are formed. The convergence of the Vid pathway with the endocytic pathway allows cells to remove intracellular and extracellular proteins simultaneously. However, the genes that regulate this step of the convergence have not been identified previously. Here we show that VID30 plays a critical role for the association of Vid vesicles and actin patches. Vid30 is constitutively expressed and interacts with Vid vesicle proteins Vid24 and Sec28 but not with the cargo protein FBPase. In the absence of SEC28 or VID24, Vid30 association with actin patches was prolonged. In cells lacking the VID30 gene, FBPase and Vid24 were not localized to actin patches, suggesting that Vid30 has a role in the association of Vid vesicles and actin patches. Vid30 contains a LisH and a CTLH domain, both of which are required for FBPase degradation. When these domains were deleted, FBPase trafficking to the vacuole was impaired. We suggest that Vid30 also has a role in the Vid pathway at a later step in a process that is mediated by the LisH and CTLH domains.  相似文献   

6.
嗜肺军团菌是一种胞内寄生菌,其通过特有的Dot/IcmType-IVB分泌系统向胞浆内分泌大量效应因子,其中已知参与宿主泛素化调控的效应因子有十多种.这些效应因子通过对宿主泛素化途径进行调控来达到逃避宿主免疫系统"监视"并大量增殖的目的.参与调控宿主泛素化途径的效应因子包括 AnkB、SidC、LubX、SidH、Le...  相似文献   

7.
Eukaryotic cells can employ autophagy to defend themselves against invading pathogens. Upon infection by Plasmodium berghei sporozoites, the host hepatocyte targets the invader by labelling the parasitophorous vacuole membrane (PVM) with the autophagy marker protein LC3. Until now, it has not been clear whether LC3 recruitment to the PVM is mediated by fusion of autophagosomes or by direct incorporation. To distinguish between these possibilities, we knocked out genes that are essential for autophagosome formation and for direct LC3 incorporation into membranes. The CRISPR/Cas9 system was employed to generate host cell lines deficient for either FIP200, a member of the initiation complex for autophagosome formation, or ATG5, responsible for LC3 lipidation and incorporation of LC3 into membranes. Infection of these knockout cell lines with Pberghei sporozoites revealed that LC3 recruitment to the PVM indeed depends on functional ATG5 and the elongation machinery, but not on FIP200 and the initiation complex, suggesting a direct incorporation of LC3 into the PVM. Importantly, in Pberghei‐infected ATG5?/? host cells, lysosomes still accumulated at the PVM, indicating that the recruitment of lysosomes follows an LC3‐independent pathway.  相似文献   

8.
We have evaluated the fate of misfolded protein domains in the Saccharomyces cerevisiae secretory pathway by fusing mutant forms of the NH2-terminal domain of lambda repressor protein to the secreted protein invertase. The hybrid protein carrying the wild-type repressor domain is mostly secreted to the cell surface, whereas hybrid proteins with amino acid substitutions that cause the repressor domain to be thermodynamically unstable are retained intracellularly. Surprisingly, the retained hybrids are found in the vacuole, where the repressor moiety is degraded by vacuolar proteases. The following observations indicate that receptor-mediated recognition of the mutant repressor domain in the Golgi lumen targets these hybrid fusions to the vacuole. (a) The invertase-repressor fusions, like wild-type invertase, behave as soluble proteins in the ER lumen. (b) Targeting to the vacuole is saturable since overexpression of the hybrids carrying mutant repressor increases the fraction of fusion protein that appears at the cell surface. (c) Finally, deletion of the VPS10 gene, which encodes the transmembrane Golgi receptor responsible for targeting carboxypeptidase Y to the vacuole, causes the mutant hybrids to be diverted to the cell surface. Together these findings suggest that yeast have a salvage pathway for degradation of nonnative luminal proteins by receptor- mediated transport to the vacuole.  相似文献   

9.
To identify host proteins involved in Legionella pneumophila intracellular replication, the soil amoeba Dictyostelium discoideum was analysed. The absence of the amoebal RtoA protein is demonstrated here to depress L. pneumophila intracellular growth. Uptake of L. pneumophila into a D. discoideum rtoA(-) strain was marginally defective, but this effect was not sufficient to account for the defective intracellular growth of L. pneumophila. The rtoA mutant was also more resistant to high-multiplicity killing by the bacterium. A targeting assay testing the colocalization of L. pneumophila-containing vacuole with an endoplasmic reticulum/pre-Golgi intermediate compartment marker protein, GFP-HDEL, was used to analyse these defects. In parental D. discoideum, the L. pneumophila vacuole showed recruitment of GFP-HDEL within 40 min after introduction of bacteria to the amoebae. By 6 h after infection it was clear that the rtoA mutant acquired and retained the GFP-HDEL less efficiently than the parental strain, and that the mutant was defective for promoting the physical expansion of the membranous compartment surrounding the bacteria. Depressed intracellular growth of L. pneumophila in a D. discoideum rtoA(-) mutant therefore appeared to result from a lowered efficiency of vesicle trafficking events that are essential for the modification and expansion of the L. pneumophila-containing compartment.  相似文献   

10.
11.
12.
Induction of retrograde trafficking of mannosidase II and TGN38 in NRK cells and inhibition of alpha-glucosidase in vitro by nordihydroguaiaretic acid (NDGA) were strongly interfered with by serum, serum albumin, or other unrelated proteins added to the medium or incubation mixture. These observations indicate that NDGA interacts with diverse kinds of proteins, and therefore, pharmacological effects of NDGA at cellular levels should be carefully interpreted.  相似文献   

13.
Xu X  Gupta S  Hu W  McGrath BC  Cavener DR 《PloS one》2011,6(8):e23740

Background

The ER chaperone GRP78/BiP is a homolog of the Hsp70 family of heat shock proteins, yet GRP78/BiP is not induced by heat shock but instead by ER stress. However, previous studies had not considered more physiologically relevant temperature elevation associated with febrile hyperthermia. In this report we examine the response of GRP78/BiP and other components of the ER stress pathway in cells exposed to 40°C.

Methodology

AD293 cells were exposed to 43°C heat shock to confirm inhibition of the ER stress response genes. Five mammalian cell types, including AD293 cells, were then exposed to 40°C hyperthermia for various time periods and induction of the ER stress pathway was assessed.

Principal Findings

The inhibition of the ER stress pathway by heat shock (43°C) was confirmed. In contrast cells subjected to more mild temperature elevation (40°C) showed either a partial or full ER stress pathway induction as determined by downstream targets of the three arms of the ER stress pathway as well as a heat shock response. Cells deficient for Perk or Gcn2 exhibit great sensitivity to ER stress induction by hyperthermia.

Conclusions

The ER stress pathway is induced partially or fully as a consequence of hyperthermia in parallel with induction of Hsp70. These findings suggest that the ER and cytoplasm of cells contain parallel pathways to coordinately regulate adaptation to febrile hyperthermia associated with disease or infection.  相似文献   

14.
The gluconeogenic enzyme fructose-1,6-bisphosphatase (FBPase) is degraded in the vacuole when glucose is added to glucose-starved cells. Before it is delivered to the vacuole, however, FBPase is imported into intermediate carriers called Vid (vacuole import and degradation) vesicles. Here, using biochemical and genetic approaches, we identified a requirement for SEC28 in FBPase degradation. SEC28 encodes the epsilon-COP subunit of COPI (coat protein complex I) coatomer proteins. When SEC28 and other coatomer genes were mutated, FBPase degradation was defective and FBPase association with Vid vesicles was impaired. Coatomer proteins were identified as components of Vid vesicles, and they formed a protein complex with a Vid vesicle-specific protein, Vid24p. Furthermore, Vid24p association with Vid vesicles was impaired when coatomer genes were mutated. Kinetic studies indicated that Sec28p traffics to multiple locations. Sec28p was in Vid vesicles, endocytic compartments, and the vacuolar membrane in various mutants that block the FBPase degradation pathway. Sec28p was also found in vesicles adjacent to the vacuolar membrane in the ret2-1 coatomer mutant. We propose that Sec28p resides in Vid vesicles, and these vesicles converge with the endocytic pathway. After fusion, Sec28p is distributed on the vacuolar membrane, where it concentrates on vesicles that pinch off from this organelle. FBPase also utilizes the endocytic pathway for transport to the vacuole, as demonstrated by its presence in endocytic compartments in the Deltavph1 mutant. Taken together, our results indicate a strong connection between the Vid trafficking pathway and the endocytic pathway.  相似文献   

15.
16.
《Molecular cell》2021,81(18):3866-3876.e2
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17.
The intracellular protozoan Toxoplasma gondii is auxotrophic for low-density lipoprotein (LDL)-derived cholesterol (C). We previously showed that T. gondii scavenges this essential lipid from host endolysosomal compartments and that C delivery to the parasitophorous vacuole (PV) does not require transit through host Golgi or endoplasmic reticulum. In this study, we explore the itinerary of C from the host endolysosomes to the PV. Labeled C incorporated into LDL is rapidly detected in intravacuolar parasites and partially esterified by the parasites. In contrast to diverse mammalian organelles, the post-endolysosomal transfer of C to the PV does not involve the host plasma membrane as an intermediate. Nevertheless, the PV membrane is accessible to extracellular sterol acceptors, suggesting C trafficking from intracellular parasites to host plasma membrane. C movement to the PV requires temperatures permissive for vesicular transport, metabolic energy and functional microtubules. Host caveolae vesicles and the sterol carrier protein-2 do not participate in this process. Proteolytic treatment of purified PV or free parasites abolishes C acquisition by the parasites. Altogether, these results support a vesicular transport system from host endolysosomes to the PV, and a requirement for PV membrane and parasite plasma membrane proteins in C delivery to T. gondii.  相似文献   

18.
Organelle contact sites perform fundamental functions in cells, including lipid and ion homeostasis, membrane dynamics, and signaling. Using a forward proteomics approach in yeast, we identified new ER–mitochondria and ER–vacuole contacts specified by an uncharacterized protein, Ylr072w. Ylr072w is a conserved protein with GRAM and VASt domains that selectively transports sterols and is thus termed Ltc1, for Lipid transfer at contact site 1. Ltc1 localized to ER–mitochondria and ER–vacuole contacts via the mitochondrial import receptors Tom70/71 and the vacuolar protein Vac8, respectively. At mitochondria, Ltc1 was required for cell viability in the absence of Mdm34, a subunit of the ER–mitochondria encounter structure. At vacuoles, Ltc1 was required for sterol-enriched membrane domain formation in response to stress. Increasing the proportion of Ltc1 at vacuoles was sufficient to induce sterol-enriched vacuolar domains without stress. Thus, our data support a model in which Ltc1 is a sterol-dependent regulator of organelle and cellular homeostasis via its dual localization to ER–mitochondria and ER–vacuole contact sites.  相似文献   

19.
The proper functioning of eukaryotic organelles is largely dependent on the specific packaging of cargo proteins within transient delivery vesicles. The cytoplasm to vacuole targeting (Cvt) pathway is an autophagy-related trafficking pathway whose cargo proteins, aminopeptidase I and alpha-mannosidase, are selectively transported from the cytoplasm to the lysosome-like vacuole in yeast. This study elucidates a molecular mechanism for cargo specificity in this pathway involving four discrete steps. The Cvt19 receptor plays a central role in this process: distinct domains in Cvt19 recognize oligomerized cargo proteins and link them to the vesicle formation machinery via interaction with Cvt9 and Aut7. Because autophagy is the primary mechanism for organellar turnover, these results offer insights into physiological processes that are critical in cellular homeostasis, including specific packaging of damaged or superfluous organelles for lysosomal delivery and breakdown.  相似文献   

20.
Encephalitozoon microsporidia proliferate and differentiate within a parasitophorous vacuole. Using the fluorescent probe, calcein, and the mitochondrial probe, MitoTracker-CMXRos, a vital method was developed that confirmed ultrastructural reports that the host cell mitochondria frequently lie in immediate proximity to the parasitophorous vacuole. Morphometry failed to demonstrate any infection-induced increase in host cell mitochondria as there was no correlation between the mitochondrial volume and the extent of infection as judged by the parasitophorous vacuole volume. The total ATP concentration of infected cells did not differ from that of uninfected cells in spite of the increased metabolic demands of the infection. Treatment with 10(-6) M albendazole, more than ten times the antiparasitic IC50 dose, and demecolcine had no subjective effect on the proximity of mitochondria to the parasitophorous vacuole membrane when studied by either transmission electron microscopy or by confocal microscopy even though these drug concentrations affected microtubule structure. Thus, once the association between mitochondria and the parasitophorous vacuole has been established, host cell microtubule integrity is probably not required for its maintenance. It is unlikely that the antimicrosporidial action of albendazole involves physically uncoupling developing parasite stages from host cell organelle metabolic support.  相似文献   

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