Phosphatidylglycerol (PG) an important membrane phospholipid required for the synthesis of diphos-phatidylglycerol (DPG) commonly known as cardiolipin (CL) was identified in the fraction of endo-plasmic reticulum (ER)...Phosphatidylglycerol (PG) an important membrane phospholipid required for the synthesis of diphos-phatidylglycerol (DPG) commonly known as cardiolipin (CL) was identified in the fraction of endo-plasmic reticulum (ER)-derived transport vesicles which had no affinity for Golgi. The vesicles were produced in the presence of Brefeldin A (BFA), the agent known to inhibit ER-Golgi transport, and found to display affinity to mitochondria. The analysis revealed that their cargo was not containing proteins that are transported to Golgi, and that their membrane was free of phosphatidylinositol (PI) and ceramides (Cer). The incubation of PG-containing transport vesicles with mitochondria afforded incorporation of their membrane into the Outer Mito-chondrial Membrane (OMM) and formation of lyso-phosphatidylglycerol (LPG). In turn, upon further incubation with fresh transport active cytosol, the mitochondrial LPG was converted to PG. The results of analysis of the OMM, Inner Mitochondrial Mem-brane (IMM) and Inner Mitochondrial Space Components (IMSC) strongly suggest that PG-containing transport vesicles deliver nuclear DNA translation products to the IMSC and thus facilitate CL synthesis in the IMM. In summary, our studies provide evidence that ER-generated PG-enriched transport vesicles represent the general pathway for restitution of mitochondrial membranes and the delivery of nuclear DNA translation products that generate CL, and thus sustain the mitochondrial matrix CL-dependent metabolic reactions.展开更多
The autophagosomes were identified in the viable cycloheximide (CHX)-treated cells which had an incapacitated translational process and thus disabled synthesis of endoplasmic reticulum (ER)-derived vesicular transport...The autophagosomes were identified in the viable cycloheximide (CHX)-treated cells which had an incapacitated translational process and thus disabled synthesis of endoplasmic reticulum (ER)-derived vesicular transporters. They were found devoid of the proteins transported from ER to cell organelles, were unable to fuse with ER, Golgi or mitochondria, and displayed affinity with lysosomes. The analysis of autophagosomes, derived from the CHX cell organelles, revealed that their lipid composition resemble that of the maternal organelle. Thus, the ER-derived autophagosomes were marked with the presence of phosphatidylinositol (PI), Golgi-derived vesicles contained sphingomyelin (SM) and glycosphingolipids (GLL), and the mitochondria-derived autophagosomes contained phosphatidylglycerol (PG) and cardiolipin (CL). The incubation of the vesicles with intact lysosomes afforded their and the lysosome membrane lipids degradation. The analysis of the products derived from incubation of lysosomes and autophagosomes with radiolabeled SM, in the presence and the absence of TritonX100, allowed us to conclude that during autophagosome degradation the lysosomal enzymes are not released to cytosol, and that only lysosomes contain the enzymes degrading membrane lipids. In summary, our findings allowed us to authenticate the vesicles generated in the CHX-treated cells as organelle-specific autophagosomes and to determine that complete cycle of cell restitution and debridement includes intralysosomal degradation of the lysosomal membrane engulfing the autophagosomes vesicles.展开更多
The processes of mitochondrial restitution are controlled by nuclear genes that encode proteins synthesized in ER and cytosol and delivered as organelle- and membrane-specific transport vesicles. The analysis of the t...The processes of mitochondrial restitution are controlled by nuclear genes that encode proteins synthesized in ER and cytosol and delivered as organelle- and membrane-specific transport vesicles. The analysis of the transporters recovered from inner mitochondrial space (Mitosol) revealed that the ER-synthesized mitochondria-specific transport vesicles consist of two carriers, one remaining in outer mitochondrial membrane (OMM), and the other that transfers specific membrane segments to the inner mitochondrial membrane (IMM). The ER-assembled and IMM-committed membrane segments, while first integrated into OMM, undergo intra-mitochondrial lipid modification reflected in the synthesis of cardiolipin (CL) and inversion into Mitosol with load of IMM associated cytosolic proteins. Then, the CL-bedecked vesicles are released from OMM to Mitosol and upon contact with IMM fuse with the membrane, and the release of cytosolic cargo ensues. While ER-assembled mitochondria-specific transport vesicles fuse with OMM with the aid of the cytosolic, phosphatidylglycerol (PG)-specific phospholipase A2 (PLA2), the Mitosol-contained CL-specific PLA guides vesicles fusion with IMM. The described path of translocation of the membrane segments and the cytosol synthesized proteins into the designated mitochondrial compartments sustains growth and identity of OMM, IMM, maintains protein delivery for intra-mitochondrial lipid and protein modification in Mitosol, and ensures conformity of the cytosolic proteins cargo delivered to matrix.展开更多
Does the ER subdomain that associates with the chloroplast in vivo, hereafter referred to as the chloroplast/ER nexus, play a role in protein flow within the ER？ In studies of tobacco cells either constitutively or t...Does the ER subdomain that associates with the chloroplast in vivo, hereafter referred to as the chloroplast/ER nexus, play a role in protein flow within the ER？ In studies of tobacco cells either constitutively or transiently expressing ER-retained luminal, GFP-HDEL, or trans-membrane, YFP-RHD3, fluorescent fusion proteins, brief 405-nm （3-6-mW） laser stimulation of the nexus causes a qualitative difference in the movement and behavior of proteins in the ER. Photostimulating the nexus produces fluorescent protein punctate aggregates （boluses） within the lumen and membrane of the ER. The aggregation propagates through the membrane network throughout the cell, but within minutes can revert to normal, with disaggregation propagating back toward the originally photostimulated nexus. In the meantime, the ER grows and anastomoses around the chloroplast, forming a dense cisternal and tubular network. If this network is again photostimulated, bolus formation does not recur and, if the photostimulation results in photobleaching, fluorescence recovery after photobleaching occurs as it would typically in areas away from the nexus. Bolus propagation is not mediated by the actin cytoskeleton, but can be reversed by pre-conditioning the cells for 30 min with high, 40-45℃, temperature （heat stress）. Because it is not reversed with heat stress, the reorganization of the ER at the nexus following photostimulation is a separate event.展开更多
The nucleus-initiated augmentation of ER membrane is reflected in a coordinated synthesis and intercalation of the explicit proteins and lipids required for the replacement, repair and function of the cell and its org...The nucleus-initiated augmentation of ER membrane is reflected in a coordinated synthesis and intercalation of the explicit proteins and lipids required for the replacement, repair and function of the cell and its organelles. The direct connection between nucleus and the membranes containing labeled sphingosine (SphN) and ceramide (Cer) was affirmed by determining synthetic activity of serine palmitoyltransferase (SPT). The SPT and the newly synthesized serine-labeled lipid products were identified in the Outer- and Inner-Nuclear Membrane (ONM, INM) and ER. The pulse-chase experiments disclosed that the incorporation of radiolabeled lipids into both nuclear membranes declined upon their simultaneous increase in Endoplasmic Reticulum (ER). These results, and prior findings regarding metabolic transfer of nuclear membrane phosphoinositides to the outer leaflet of ER [Slomiany and Slomiany, Health, 2011, 3, 187-199], allowed us to reason that INM and ONM are not distinct entities, but uninterrupted continuum facing nucleosol and then cytosol when protracted into segment known as ER. Consequently, the identification of SPT and its products in the inner leaflet of nuclear and ER microsomes lent credence to the luminal presence of Cer in Golgi, luminal synthesis of glycosphingolipids (GSphLs), sphingomyelin (SM), and their delivery to the outer leaflet of apical and basolateral cell membrane, respectively. The findings presented in this communication provide further support to our concept that the factual intercalation of proteins and lipids into the cell membranes can only take place during their simultaneous synthesis that is guided by the nuclear and cytosolic processes enacted in nuclear-ER membrane continuum. At the nuclear stage, the signal-specific genes expression promotes active synthesis and intercalation of lipids into the organelles’ customized membrane that is protracted and articulated in ER in form of transport vesicles.展开更多
Restitution of the cell organelles and the membrane implicates serine palmitoyltransferase (SPT) in signal-specific and selective assembly of the transport vesicles. Here, we reveal that SPT, embedded in the outer lea...Restitution of the cell organelles and the membrane implicates serine palmitoyltransferase (SPT) in signal-specific and selective assembly of the transport vesicles. Here, we reveal that SPT, embedded in the outer leaflet (OL) of endoplasmic reticulum (ER), is engaged in the synthesis of ER transport vesicles that recondition cell organelles, and the inner leaflet (IL) SPT in the restitution of the cell membrane. The OL SPT impacts assembly of sphingomyelinase (SMase)—susceptible ER vesicles but not the SMase-resistant and sphingolipid (SPhL) core—carrying vesicles that refurbish the cell membrane. The investigation of the SPT-initiated differences in the placement of SPhL in vesicular membranes by utilizing ER depleted of OL SPT, allows us to conclude that the restitution of endosomal and lysosomal membranes is achieved with the involvement of OL SPT, whereas the IL SPT is involved in formation of the lipid core for glycosphingolipids (GSL) and sphingomyelin (SM) of the apical and basolateral cell membrane. These findings along with our previously published report (Slomiany and Slomiany, Advances in Biological Chemistry, 2013, 3, 275-287), provide a clear distinction between the processes that renovate cell membrane and its organelles from that of the endocytotic cell debridement, and show that vesicles are navigated to the specific organelles and the cell membrane by the biomembrane constituents programmed in ER.展开更多
文摘Phosphatidylglycerol (PG) an important membrane phospholipid required for the synthesis of diphos-phatidylglycerol (DPG) commonly known as cardiolipin (CL) was identified in the fraction of endo-plasmic reticulum (ER)-derived transport vesicles which had no affinity for Golgi. The vesicles were produced in the presence of Brefeldin A (BFA), the agent known to inhibit ER-Golgi transport, and found to display affinity to mitochondria. The analysis revealed that their cargo was not containing proteins that are transported to Golgi, and that their membrane was free of phosphatidylinositol (PI) and ceramides (Cer). The incubation of PG-containing transport vesicles with mitochondria afforded incorporation of their membrane into the Outer Mito-chondrial Membrane (OMM) and formation of lyso-phosphatidylglycerol (LPG). In turn, upon further incubation with fresh transport active cytosol, the mitochondrial LPG was converted to PG. The results of analysis of the OMM, Inner Mitochondrial Mem-brane (IMM) and Inner Mitochondrial Space Components (IMSC) strongly suggest that PG-containing transport vesicles deliver nuclear DNA translation products to the IMSC and thus facilitate CL synthesis in the IMM. In summary, our studies provide evidence that ER-generated PG-enriched transport vesicles represent the general pathway for restitution of mitochondrial membranes and the delivery of nuclear DNA translation products that generate CL, and thus sustain the mitochondrial matrix CL-dependent metabolic reactions.
文摘The autophagosomes were identified in the viable cycloheximide (CHX)-treated cells which had an incapacitated translational process and thus disabled synthesis of endoplasmic reticulum (ER)-derived vesicular transporters. They were found devoid of the proteins transported from ER to cell organelles, were unable to fuse with ER, Golgi or mitochondria, and displayed affinity with lysosomes. The analysis of autophagosomes, derived from the CHX cell organelles, revealed that their lipid composition resemble that of the maternal organelle. Thus, the ER-derived autophagosomes were marked with the presence of phosphatidylinositol (PI), Golgi-derived vesicles contained sphingomyelin (SM) and glycosphingolipids (GLL), and the mitochondria-derived autophagosomes contained phosphatidylglycerol (PG) and cardiolipin (CL). The incubation of the vesicles with intact lysosomes afforded their and the lysosome membrane lipids degradation. The analysis of the products derived from incubation of lysosomes and autophagosomes with radiolabeled SM, in the presence and the absence of TritonX100, allowed us to conclude that during autophagosome degradation the lysosomal enzymes are not released to cytosol, and that only lysosomes contain the enzymes degrading membrane lipids. In summary, our findings allowed us to authenticate the vesicles generated in the CHX-treated cells as organelle-specific autophagosomes and to determine that complete cycle of cell restitution and debridement includes intralysosomal degradation of the lysosomal membrane engulfing the autophagosomes vesicles.
文摘The processes of mitochondrial restitution are controlled by nuclear genes that encode proteins synthesized in ER and cytosol and delivered as organelle- and membrane-specific transport vesicles. The analysis of the transporters recovered from inner mitochondrial space (Mitosol) revealed that the ER-synthesized mitochondria-specific transport vesicles consist of two carriers, one remaining in outer mitochondrial membrane (OMM), and the other that transfers specific membrane segments to the inner mitochondrial membrane (IMM). The ER-assembled and IMM-committed membrane segments, while first integrated into OMM, undergo intra-mitochondrial lipid modification reflected in the synthesis of cardiolipin (CL) and inversion into Mitosol with load of IMM associated cytosolic proteins. Then, the CL-bedecked vesicles are released from OMM to Mitosol and upon contact with IMM fuse with the membrane, and the release of cytosolic cargo ensues. While ER-assembled mitochondria-specific transport vesicles fuse with OMM with the aid of the cytosolic, phosphatidylglycerol (PG)-specific phospholipase A2 (PLA2), the Mitosol-contained CL-specific PLA guides vesicles fusion with IMM. The described path of translocation of the membrane segments and the cytosol synthesized proteins into the designated mitochondrial compartments sustains growth and identity of OMM, IMM, maintains protein delivery for intra-mitochondrial lipid and protein modification in Mitosol, and ensures conformity of the cytosolic proteins cargo delivered to matrix.
文摘Does the ER subdomain that associates with the chloroplast in vivo, hereafter referred to as the chloroplast/ER nexus, play a role in protein flow within the ER？ In studies of tobacco cells either constitutively or transiently expressing ER-retained luminal, GFP-HDEL, or trans-membrane, YFP-RHD3, fluorescent fusion proteins, brief 405-nm （3-6-mW） laser stimulation of the nexus causes a qualitative difference in the movement and behavior of proteins in the ER. Photostimulating the nexus produces fluorescent protein punctate aggregates （boluses） within the lumen and membrane of the ER. The aggregation propagates through the membrane network throughout the cell, but within minutes can revert to normal, with disaggregation propagating back toward the originally photostimulated nexus. In the meantime, the ER grows and anastomoses around the chloroplast, forming a dense cisternal and tubular network. If this network is again photostimulated, bolus formation does not recur and, if the photostimulation results in photobleaching, fluorescence recovery after photobleaching occurs as it would typically in areas away from the nexus. Bolus propagation is not mediated by the actin cytoskeleton, but can be reversed by pre-conditioning the cells for 30 min with high, 40-45℃, temperature （heat stress）. Because it is not reversed with heat stress, the reorganization of the ER at the nexus following photostimulation is a separate event.
文摘The nucleus-initiated augmentation of ER membrane is reflected in a coordinated synthesis and intercalation of the explicit proteins and lipids required for the replacement, repair and function of the cell and its organelles. The direct connection between nucleus and the membranes containing labeled sphingosine (SphN) and ceramide (Cer) was affirmed by determining synthetic activity of serine palmitoyltransferase (SPT). The SPT and the newly synthesized serine-labeled lipid products were identified in the Outer- and Inner-Nuclear Membrane (ONM, INM) and ER. The pulse-chase experiments disclosed that the incorporation of radiolabeled lipids into both nuclear membranes declined upon their simultaneous increase in Endoplasmic Reticulum (ER). These results, and prior findings regarding metabolic transfer of nuclear membrane phosphoinositides to the outer leaflet of ER [Slomiany and Slomiany, Health, 2011, 3, 187-199], allowed us to reason that INM and ONM are not distinct entities, but uninterrupted continuum facing nucleosol and then cytosol when protracted into segment known as ER. Consequently, the identification of SPT and its products in the inner leaflet of nuclear and ER microsomes lent credence to the luminal presence of Cer in Golgi, luminal synthesis of glycosphingolipids (GSphLs), sphingomyelin (SM), and their delivery to the outer leaflet of apical and basolateral cell membrane, respectively. The findings presented in this communication provide further support to our concept that the factual intercalation of proteins and lipids into the cell membranes can only take place during their simultaneous synthesis that is guided by the nuclear and cytosolic processes enacted in nuclear-ER membrane continuum. At the nuclear stage, the signal-specific genes expression promotes active synthesis and intercalation of lipids into the organelles’ customized membrane that is protracted and articulated in ER in form of transport vesicles.
文摘Restitution of the cell organelles and the membrane implicates serine palmitoyltransferase (SPT) in signal-specific and selective assembly of the transport vesicles. Here, we reveal that SPT, embedded in the outer leaflet (OL) of endoplasmic reticulum (ER), is engaged in the synthesis of ER transport vesicles that recondition cell organelles, and the inner leaflet (IL) SPT in the restitution of the cell membrane. The OL SPT impacts assembly of sphingomyelinase (SMase)—susceptible ER vesicles but not the SMase-resistant and sphingolipid (SPhL) core—carrying vesicles that refurbish the cell membrane. The investigation of the SPT-initiated differences in the placement of SPhL in vesicular membranes by utilizing ER depleted of OL SPT, allows us to conclude that the restitution of endosomal and lysosomal membranes is achieved with the involvement of OL SPT, whereas the IL SPT is involved in formation of the lipid core for glycosphingolipids (GSL) and sphingomyelin (SM) of the apical and basolateral cell membrane. These findings along with our previously published report (Slomiany and Slomiany, Advances in Biological Chemistry, 2013, 3, 275-287), provide a clear distinction between the processes that renovate cell membrane and its organelles from that of the endocytotic cell debridement, and show that vesicles are navigated to the specific organelles and the cell membrane by the biomembrane constituents programmed in ER.
