Evidence for an ER to Golgi to chloroplast protein transport pathway

Chloroplast protein import is generally believed to occur posttranslationally through the interaction of a precursor protein with the Toc and Tic transport apparatus in the plastid envelope membranes. The cleavable N-terminal transit peptide present on translocated proteins has been considered to be essential and sufficient for targeting. This idea was recently challenged when an analysis of the chloroplast proteome revealed many proteins without a predicted transit peptide. A recent study demonstrates the existence of a novel chloroplast targeting pathway, starting with protein entry into the endoplasmic reticulum and involving the Golgi apparatus.     

Reconstitution of GTP-binding Sar1 protein function in ER to Golgi transport

In the yeast secretory pathway, two genes SEC12 and SAR1, which encode a 70-kD integral membrane protein and a 21-kD GTP-binding protein, respectively, cooperate in protein transport from the ER to the Golgi apparatus. In vivo, the elevation of the SAR1 dosage suppresses temperature sensitivity of the sec12 mutant. In this paper, we show cell-free reconstitution of the ER-to-Golgi transport that depends on both of these gene products. First, the membranes from the sec12 mutant cells reproduce temperature sensitivity in the in vitro ER-to-Golgi transport reaction. Furthermore, the addition of the Sar1 protein completely suppresses this temperature-sensitive defect of the sec12 membranes. The analysis of Sar1p partially purified by E. coli expression suggests that GTP hydrolysis is essential for Sar1p to execute its function.     

Dynamics of Golgi matrix proteins after the blockage of ER to Golgi transport

When the ER to Golgi transport is blocked by a GTP-restricted mutant of Sar1p (H79G) in NRK-52E cells, most Golgi resident proteins are transported back into the ER. In contrast, the cis-Golgi matrix proteins GM130 and GRASP65 are retained in punctate cytoplasmic structures, namely Golgi remnants. Significant amounts of the medial-Golgi matrix proteins golgin-45, GRASP55 and giantin are retained in the Golgi remnants, but a fraction of these proteins relocates to the ER. Golgin-97, a candidate trans-Golgi network matrix protein, is retained in Golgi remnant-like structures, but mostly separated from GM130 and GRASP65. Interestingly, most Sec13p, a COPII component, congregates into larger cytoplasmic clusters soon after the microinjection of Sar1p(H79G), and these move to accumulate around the Golgi apparatus. Sec13p clusters remain associated with Golgi remnants after prolonged incubation. Electron microscopic analysis revealed that Golgi remnants are clusters of larger vesicles with smaller vesicles, many of which are coated. GM130 is mainly associated with larger vesicles and Sec13p with smaller coated vesicles. The Sec13p clusters disperse when p115 binding to the Golgi apparatus is inhibited. These results suggest that cis-Golgi matrix proteins resist retrograde transport flow and stay as true residents in Golgi remnants after the inhibition of ER to Golgi transport.     

Ilimaquinone inhibits gap-junctional communication prior to Golgi fragmentation and block in protein transport

Brefeldin A and ilimaquinone are compounds known to affect Golgi structure and function. In particular, the transport of proteins is blocked either at the level of exit from endoplasmic reticulum (brefeldin) or at cis-Golgi (ilimaquinone). Brefeldin caused a slow decrease in gap-junctional communication and a slow loss of all phosphorylated forms of connexin43 in hamster and rat fibroblasts, while ilimaquinone caused an abrupt decrease in gap-junctional communication and rapid loss of only the slowest migrating phosphorylated connexin43 band (P2). Ilimaquinone caused these effects prior to any significant Golgi fragmentation, especially in hamster fibroblasts. Concurrently, ilimaquinone minimally affected protein secretion, while brefeldin caused an instantaneous decrease. These results show that ilimaquinone inhibits gap-junctional communication in connexin43-expressing cells by a mechanism not dependent on Golgi fragmentation or block in protein transport.     

ER to Golgi transport: Requirement for p115 at a pre-Golgi VTC stage

The membrane transport factor p115 functions in the secretory pathway of mammalian cells. Using biochemical and morphological approaches, we show that p115 participates in the assembly and maintenance of normal Golgi structure and is required for ER to Golgi traffic at a pre-Golgi stage. Injection of antibodies against p115 into intact WIF-B cells caused Golgi disruption and inhibited Golgi complex reassembly after BFA treatment and wash-out. Addition of anti-p115 antibodies or depletion of p115 from a VSVtsO45 based semi-intact cell transport assay inhibited transport. The inhibition occurred after VSV glycoprotein (VSV-G) exit from the ER but before its delivery to the Golgi complex, and resulted in VSV-G protein accumulating in peripheral vesicular tubular clusters (VTCs). The p115-requiring step of transport followed the rab1-requiring step and preceded the Ca(2+)-requiring step. Unexpectedly, mannosidase I redistributed from the Golgi complex to colocalize with VSV-G protein arrested in pre-Golgi VTCs by p115 depletion. Redistribution of mannosidase I was also observed in cells incubated at 15 degrees C. Our data show that p115 is essential for the translocation of pre-Golgi VTCs from peripheral sites to the Golgi stack. This defines a previously uncharacterized function for p115 at the VTC stage of ER to Golgi traffic.     

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.     

Transforming ER exit: protein secretion meets oncogenesis

COPII-coated vesicles drive protein export from the endoplasmic reticulum (ER), although the regulation of this event, both spatially and kinetically, remains unclear. TFG is now defined as a factor that modulates recruitment of the coat and links ER sequestration of kinases to oncogenesis.     

Identification of a novel ADAMTS9/GON-1 function for protein transport from the ER to the Golgi

A disintegrin-like and metalloprotease with thrombospondin type I motif (ADAMTS9) is a member of the secreted metalloprotease family that is believed to digest extracellular matrix (ECM) proteins outside of cells. Its Caenorhabditis elegans orthologue, GON-1, is involved in ECM degradation and is required for gonad morphogenesis. ADAMTS9 and GON-1 have similar domain structures, and both have a unique C-terminal domain called the "GON domain," whose function remains unknown. Here we show that down-regulation of human ADAMTS9 and C. elegans GON-1 results in the inhibition of protein transport from the endoplasmic reticulum (ER) to the Golgi. This phenotype was rescued by the expression of the GON domain localizing in the ER in human cells and C. elegans. We propose a novel function of ADAMTS9 and GON-1 in the ER that promotes protein transport from the ER to the Golgi. This function is GON-domain dependent but protease activity independent.     

COPI-mediated retrograde trafficking from the Golgi to the ER regulates EGFR nuclear transport

Emerging evidence indicates that cell surface receptors, such as the entire epidermal growth factor receptor (EGFR) family, have been shown to localize in the nucleus. A retrograde route from the Golgi to the endoplasmic reticulum (ER) is postulated to be involved in the EGFR trafficking to the nucleus; however, the molecular mechanism in this proposed model remains unexplored. Here, we demonstrate that membrane-embedded vesicular trafficking is involved in the nuclear transport of EGFR. Confocal immunofluorescence reveals that in response to EGF, a portion of EGFR redistributes to the Golgi and the ER, where its NH₂-terminus resides within the lumen of Golgi/ER and COOH-terminus is exposed to the cytoplasm. Blockage of the Golgi-to-ER retrograde trafficking by brefeldin A or dominant mutants of the small GTPase ADP-ribosylation factor, which both resulted in the disassembly of the coat protein complex I (COPI) coat to the Golgi, inhibit EGFR transport to the ER and the nucleus. We further find that EGF-dependent nuclear transport of EGFR is regulated by retrograde trafficking from the Golgi to the ER involving an association of EGFR with γ-COP, one of the subunits of the COPI coatomer. Our findings experimentally provide a comprehensive pathway that nuclear transport of EGFR is regulated by COPI-mediated vesicular trafficking from the Golgi to the ER, and may serve as a general mechanism in regulating the nuclear transport of other cell surface receptors.     

Morphological analysis of protein transport from the ER to Golgi membranes in digitonin-permeabilized cells: role of the P58 containing compartment

The glycoside digitonin was used to selectively permeabilize the plasma membrane exposing functionally and morphologically intact ER and Golgi compartments. Permeabilized cells efficiently transported vesicular stomatitis virus glycoprotein (VSV-G) through sealed, membrane-bound compartments in an ATP and cytosol dependent fashion. Transport was vectorial. VSV-G protein was first transported to punctate structures which colocalized with p58 (a putative marker for peripheral punctate pre-Golgi intermediates and the cis-Golgi network) before delivery to the medial Golgi compartments containing alpha-1,2-mannosidase II and processing of VSV-G to endoglycosidase H resistant forms. Exit from the ER was inhibited by an antibody recognizing the carboxyl-terminus of VSV-G. In contrast, VSV-G protein colocalized with p58 in the absence of Ca2+ or the presence of an antibody which inhibits the transport component NSF (SEC18). These studies demonstrate that digitonin permeabilized cells can be used to efficiently reconstitute the early secretory pathway in vitro, allowing a direct comparison of the morphological and biochemical events involved in vesicular tafficking, and identifying a key role for the p58 containing compartment in ER to Golgi transport.     

Vesicular and nonvesicular transport of ceramide from ER to the Golgi apparatus in yeast.

Transport and sorting of lipids must occur with specific mechanisms because the membranes of intracellular organelles differ in lipid composition even though most lipid biosynthesis begins in the ER. In yeast, ceramide is synthesized in the ER and transferred to the Golgi apparatus where inositolphosphorylceramide (IPC) is formed. These two facts imply that ceramide can be transported to the Golgi independent of vesicular traffic because IPC synthesis still continues when vesicular transport is blocked in sec mutants. Nonvesicular IPC synthesis in intact cells is not affected by ATP depletion. Using an in vitro assay that reconstitutes the nonvesicular pathway for transport of ceramide, we found that transport is temperature and cytosol dependent but energy independent. Preincubation of ER and Golgi fractions together at 4 degrees C, where ceramide transport does not occur, rendered the transport reaction membrane concentration independent, providing biochemical evidence that ER-Golgi membrane contacts stimulate ceramide transport. A cytosolic protease-sensitive factor is required after establishment of ER-Golgi contacts.     

Biogenesis of endoplasmic reticulum transport vesicles transferring gastric apomucin from ER to Golgi.

Rough endoplasmic reticulum (RER) transport vesicles were generated from gastric mucous cell RER microsomes in the presence of labeled precursors of phospholipids. The vesicles contained 7-10% of their proteins in the form of apomucin (cargo), and 80% of de novo synthesized phosphatidylcholine (PC) was incorporated into the vesicular membrane. In the absence of choline and ethanolamine precursors or in the presence of 3 mM N-ethylmaleimide (NEM), an inhibitor of CTP:phosphocholine cytidylyltransferase, formation of the transport vesicles, their enrichment in the newly synthesized PC, and the total synthesis of PC decreased by 86%, whereas in the presence of 3 mM Zn2+, complete blockage of vesicle formation and PC synthesis was observed. Analysis of the mucin-transporting vesicles indicated that the CTP:phosphocholine cytidylyltransferase and 1,2-diacyl-sn-glycerol:CDP-choline phosphotransferase remained associated with transport vesicles released from ER. The enzymes and other proteins separated from the vesicle surface prior to vesicle fusion with Golgi and the process was induced by phosphorylation. Based on the results of this study, it is proposed that the formation of the ER transport vesicles of gastric mucosal cells is in concert with synthesis of phospholipids and thus in part is regulated by phospholipid-synthesizing enzymes that reside on the membrane during its biogenesis and dissociate from its surface once the task is completed.     

Isolation of a functional vesicular intermediate that mediates ER to Golgi transport in yeast

We have used an in vitro assay that reconstitutes transport from the ER to the Golgi complex in yeast to identify a functional vesicular intermediate in transit to the Golgi apparatus. Permeabilized yeast cells, which serve as the donor in this assay, release a homogeneous population of vesicles that are biochemically distinct from the donor ER fraction. The isolated vesicles, containing a post-ER/pre-Golgi form of the marker protein pro-alpha-factor, were able to bind to and fuse with exogenously added Golgi membranes. The ability to isolate fusion competent vesicles provides direct evidence that ER to Golgi membrane transport is mediated by a discrete population of vesicular carriers.     

Cholesterol ester transfer protein and apolipoprotein E gene polymorphisms in hyperlipidemic Asian Indians in North India

We determined the distribution of the polymorphic variants of CETP TaqIB and ApoE genes and their association with lipid and anthropometric parameters in hyperlipidemic and normolipidemic Asian Indians in North India. CETP TaqIB and ApoE polymorphism were assayed by PCR-RFLP in hyperlipidemic (n = 220) and normolipidemic (n = 367) subjects. Plasma lipids levels were estimated using commercially available kits from Randox (USA). The distribution of CETP TaqIB genotypes and alleles did not differ between the two groups. The frequency of ApoE ε4 allele was significantly higher in hyperlipidemic than normolipidemic subjects. Serum lipid levels were comparable between subjects with the different CETP TaqIB and ApoE genotypes in the two groups. Multivariate analysis after adjusting for age, sex, BMI, WHR, and total skinfold thickness showed that subjects with the Ε3Ε4 genotype and ε4 allele carriers were at significantly higher odds to develop hyperlipidemia [2.07 (1.29-3.30) and 2.05 (1.30-3.24), respectively] as compared to the other genotypes. ApoE ε4 allele and E3E4 genotype emerged as important genetic markers for hyperlipidemia in this study population.     

Cholesterol can stimulate secretion of apolipoprotein B by cultured human hepatocytes

During a 5 day cultivation of human hepatocytes in a primary culture the secretion of apolipoprotein B was measured by enzyme-linked immunosorbent assay. Density-gradient ultracentrifugation demonstrated that the majority of the secreted apolipoprotein B was associated with the very-low-density lipoprotein fraction. Exposure of the cells to cholesterol (5-100 micrograms/ml) resulted in a dose-dependent increase in apolipoprotein B secretion rate.     

Degradation of endoplasmic reticulum (ER) quality control substrates requires transport between the ER and Golgi

Endoplasmic reticulum (ER) quality control (ERQC) components retain and degrade misfolded proteins, and our results have found that the degradation of the soluble ERQC substrates CPY* and PrA* but not membrane spanning ERQC substrates requires transport between the ER and Golgi. Stabilization of these misfolded soluble proteins was seen in cells lacking Erv29p, a probable Golgi localized protein that cycles through the ER by means of a di-lysine ER retrieval motif (KKKIY). Cells lacking Erv29p also displayed severely retarded ER exit kinetics for a subset of correctly folded proteins. We suggest that Erv29p is likely involved in cargo loading of a subset of proteins, including soluble misfolded proteins, into vesicles for ER exit. The stabilization of soluble ERQC substrates in both erv29Delta cells and sec mutants blocked in either ER exit (sec12) or vesicle delivery to the Golgi (sec18) suggests that ER-Golgi transport is required for ERQC and reveals a new aspect of the degradative mechanism.     

Quantitative ER <--> Golgi transport kinetics and protein separation upon Golgi exit revealed by vesicular integral membrane protein 36 dynamics in live cells

To quantitatively investigate the trafficking of the transmembrane lectin VIP36 and its relation to cargo-containing transport carriers (TCs), we analyzed a C-terminal fluorescent-protein (FP) fusion, VIP36-SP-FP. When expressed at moderate levels, VIP36-SP-FP localized to the endoplasmic reticulum, Golgi apparatus, and intermediate transport structures, and colocalized with epitope-tagged VIP36. Temperature shift and pharmacological experiments indicated VIP36-SP-FP recycled in the early secretory pathway, exhibiting trafficking representative of a class of transmembrane cargo receptors, including the closely related lectin ERGIC53. VIP36-SP-FP trafficking structures comprised tubules and globular elements, which translocated in a saltatory manner. Simultaneous visualization of anterograde secretory cargo and VIP36-SP-FP indicated that the globular structures were pre-Golgi carriers, and that VIP36-SP-FP segregated from cargo within the Golgi and was not included in post-Golgi TCs. Organelle-specific bleach experiments directly measured the exchange of VIP36-SP-FP between the Golgi and endoplasmic reticulum (ER). Fitting a two-compartment model to the recovery data predicted first order rate constants of 1.22 +/- 0.44%/min for ER --> Golgi, and 7.68 +/- 1.94%/min for Golgi --> ER transport, revealing a half-time of 113 +/- 70 min for leaving the ER and 1.67 +/- 0.45 min for leaving the Golgi, and accounting for the measured steady-state distribution of VIP36-SP-FP (13% Golgi/87% ER). Perturbing transport with AlF(4)(-) treatment altered VIP36-SP-GFP distribution and changed the rate constants. The parameters of the model suggest that relatively small differences in the first order rate constants, perhaps manifested in subtle differences in the tendency to enter distinct TCs, result in large differences in the steady-state localization of secretory components.     

Rab6 coordinates a novel Golgi to ER retrograde transport pathway in live cells

We visualized a fluorescent-protein (FP) fusion to Rab6, a Golgi-associated GTPase, in conjunction with fluorescent secretory pathway markers. FP-Rab6 defined highly dynamic transport carriers (TCs) translocating from the Golgi to the cell periphery. FP-Rab6 TCs specifically accumulated a retrograde cargo, the wild-type Shiga toxin B-fragment (STB), during STB transport from the Golgi to the endoplasmic reticulum (ER). FP-Rab6 TCs associated intimately with the ER, and STB entered the ER via specialized peripheral regions that accumulated FP-Rab6. Microinjection of antibodies that block coatomer protein I (COPI) function inhibited trafficking of a KDEL-receptor FP-fusion, but not FP-Rab6. Additionally, markers of COPI-dependent recycling were excluded from FP-Rab6/STB TCs. Overexpression of Rab6:GDP (T27N mutant) using T7 vaccinia inhibited toxicity of Shiga holotoxin, but did not alter STB transport to the Golgi or Golgi morphology. Taken together, our results indicate Rab6 regulates a novel Golgi to ER transport pathway.