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1.  Transport of soluble proteins through the Golgi occurs by diffusion via continuities across cisternae 
eLife  2014;3:e02009.
The mechanism of transport through the Golgi complex is not completely understood, insofar as no single transport mechanism appears to account for all of the observations. Here, we compare the transport of soluble secretory proteins (albumin and α1-antitrypsin) with that of supramolecular cargoes (e.g., procollagen) that are proposed to traverse the Golgi by compartment progression–maturation. We show that these soluble proteins traverse the Golgi much faster than procollagen while moving through the same stack. Moreover, we present kinetic and morphological observations that indicate that albumin transport occurs by diffusion via intercisternal continuities. These data provide evidence for a transport mechanism that applies to a major class of secretory proteins and indicate the co-existence of multiple intra-Golgi trafficking modes.
DOI: http://dx.doi.org/10.7554/eLife.02009.001
eLife digest
The Golgi is a structure within cells where proteins and other large molecules are modified and prepared for delivery to locations inside or outside of the cell. Each Golgi is made from a stack of flattened sacs called cisternae that are filled with fluid and enclosed by a membrane.
Proteins and other molecules are transported to the Golgi by packages called vesicles, which fuse with the outermost cisterna, which is known as the ‘cis-face’ of the Golgi, and unload their contents. From here, the proteins are processed and modified by enzymes as they move through the Golgi towards the ‘trans-face’ on the opposite side. The modified proteins are then re-packaged into vesicles before being sent to their intended destinations.
But how do proteins move through the Golgi? Some researchers have suggested that proteins do not actually move: rather, the stacks of the Golgi move like a conveyer belt as new cisterna are added to the cis-face. However, other researchers have proposed that molecules proceed from one cisterna to the next inside small vesicles. It is also possible that proteins are transported through the Golgi in other ways, or by a combination of two or more methods.
Now, Beznoussenko, Parashuraman et al. reveal that some small, soluble, proteins can move through the Golgi by diffusion. These proteins move much quicker than large protein complexes, which suggests that multiple transport mechanisms do co-exist within the Golgi. Furthermore, Beznoussenko, Parashuraman et al. found that these soluble proteins are most likely moving through some narrow tunnel-like connections between the individual cisternae.
Following on from the work of Beznoussenko, Parashuraman et al., the main challenge is to understand how all the different types of proteins that move through the Golgi are transported—which includes roughly a third of all human proteins. As many of these proteins are important for human health, learning to control their transport might create new opportunities to understand and treat disease.
DOI: http://dx.doi.org/10.7554/eLife.02009.002
doi:10.7554/eLife.02009
PMCID: PMC4070021  PMID: 24867214
intracellular trafficking; soluble cargo proteins; albumin; golgi complex; membrane tubules; human
2.  SLY1 and Syntaxin 18 specify a distinct pathway for procollagen VII export from the endoplasmic reticulum 
eLife  2014;3:e02784.
TANGO1 binds and exports Procollagen VII from the endoplasmic reticulum (ER). In this study, we report a connection between the cytoplasmic domain of TANGO1 and SLY1, a protein that is required for membrane fusion. Knockdown of SLY1 by siRNA arrested Procollagen VII in the ER without affecting the recruitment of COPII components, general protein secretion, and retrograde transport of the KDEL-containing protein BIP, and ERGIC53. SLY1 is known to interact with the ER-specific SNARE proteins Syntaxin 17 and 18, however only Syntaxin 18 was required for Procollagen VII export. Neither SLY1 nor Syntaxin 18 was required for the export of the equally bulky Procollagen I from the ER. Altogether, these findings reveal the sorting of bulky collagen family members by TANGO1 at the ER and highlight the existence of different export pathways for secretory cargoes one of which is mediated by the specific SNARE complex containing SLY1 and Syntaxin 18.
DOI: http://dx.doi.org/10.7554/eLife.02784.001
eLife digest
Collagens are long proteins that join individual cells together to build tissues and organs. They also provide strength and elasticity to bones, tendons, and blood vessels. Like many other proteins, collagens are produced inside cells: they are folded in a compartment called the endoplasmic reticulum, and then packaged and transported to another compartment called the Golgi. Collagens are then directed from the Golgi to their final destination, which is typically the outside of the cell.
Small proteins travel from the endoplasmic reticulum to the Golgi inside packages called vesicles. However it is not clear how large proteins like collagens are transported between these two compartments. It is known that a protein called TANGO1 is needed to direct a collagen called Procollagen VII to the outside of the cells. TANGO1 binds to Procollagen VII, and it is thought that TANGO1 delays the release of Procollagen VII from the endoplasmic reticulum, so that the vesicle can grow to a size that is able to accommodate such a bulky cargo.
Nogueira, Erlmann et al. have now discovered that TANGO1 binds to another protein called SLY1, and that this protein must also be present if Procollagen VII is to be exported from the endoplasmic reticulum. In contrast, the transport of a different type of collagen—Collagen I—does not require TANGO1 or SLY1.
SLY1 helps to fuse the membranes that enclose the structures involved in protein trafficking—such as the endoplasmic reticulum, the Golgi, and the vesicles—and this allows the cargoes of vesicles to pass from one compartment to another. Nogueira, Erlmann et al. also found that a second protein (called Syntaxin 18) is also required for the export of Procollagen VII.
Nogueira, Erlmann et al. propose that collagen VII export involves TANGO1 delaying the release of collagen from the endoplasmic reticulum so that SLY1 and Syntaxin 18 can fuse other cellular membranes to the growing transport vesicle. Following this work, the next challenge is to uncover how different types of collagens are separated from each other, and identify which specific vesicles are involved in their export.
DOI: http://dx.doi.org/10.7554/eLife.02784.002
doi:10.7554/eLife.02784
PMCID: PMC4054776  PMID: 24842878
Collagen; TANGO1; SLY1; Syntaxin 18; export; ER; human
3.  Golgi-associated RhoBTB3 targets Cyclin E for ubiquitylation and promotes cell cycle progression 
The Journal of Cell Biology  2013;203(2):233-250.
The Golgi protein RhoBTB3 in complex with CUL3 and RBX1 promotes Cyclin E ubiquitylation to allow its turnover during S phase and progression through the cell cycle.
Cyclin E regulates the cell cycle transition from G1 to S phase and is degraded before entry into G2 phase. Here we show that RhoBTB3, a Golgi-associated, Rho-related ATPase, regulates the S/G2 transition of the cell cycle by targeting Cyclin E for ubiquitylation. Depletion of RhoBTB3 arrested cells in S phase, triggered Golgi fragmentation, and elevated Cyclin E levels. On the Golgi, RhoBTB3 bound Cyclin E as part of a Cullin3 (CUL3)-dependent RING–E3 ubiquitin ligase complex comprised of RhoBTB3, CUL3, and RBX1. Golgi association of this complex was required for its ability to catalyze Cyclin E ubiquitylation and allow normal cell cycle progression. These experiments reveal a novel role for a Ras superfamily member in catalyzing Cyclin E turnover during S phase, as well as an unexpected, essential role for the Golgi as a ubiquitylation platform for cell cycle control.
doi:10.1083/jcb.201305158
PMCID: PMC3812982  PMID: 24145166
4.  ERK8 is a negative regulator of O-GalNAc glycosylation and cell migration 
eLife  2014;3:e01828.
ER O-glycosylation can be induced through relocalisation GalNAc-Transferases from the Golgi. This process markedly stimulates cell migration and is constitutively activated in more than 60% of breast carcinomas. How this activation is achieved remains unclear. Here, we screened 948 signalling genes using RNAi and imaging. We identified 12 negative regulators of O-glycosylation that all control GalNAc-T sub-cellular localisation. ERK8, an atypical MAPK with high basal kinase activity, is a strong hit and is partially localised at the Golgi. Its inhibition induces the relocation of GalNAc-Ts, but not of KDEL receptors, revealing the existence of two separate COPI-dependent pathways. ERK8 down-regulation, in turn, activates cell motility. In human breast and lung carcinomas, ERK8 expression is reduced while ER O-glycosylation initiation is hyperactivated. In sum, ERK8 appears as a constitutive brake on GalNAc-T relocalisation, and the loss of its expression could drive cancer aggressivity through increased cell motility.
DOI: http://dx.doi.org/10.7554/eLife.01828.001
eLife digest
The likelihood of an individual being able to recover from cancer depends on: where the cancer is within the body, how quickly the disease is detected and how quickly treatment is started. Cancers that have spread from their original location to another part of the body are particular challenging to treat, and cause the vast majority of cancer deaths every year.
Treatments that can recognize and eradicate cancer cells, while leaving nearby healthy cells untouched, are still needed—and so there has been a lot of research into identifying the key differences between healthy cells and cancer cells. For several decades, researchers have been aware that cancer cells have more proteins coated with modified sugars on their cell surfaces than healthy cells. This is caused by the enzymes that add these sugars to the proteins relocating from one location within the cell, the Golgi apparatus, to another, called the endoplasmic reticulum. These specific ‘sugar-coated’ proteins are known to encourage cancer cells to migrate and invade new tissues, but the mechanisms that regulate the addition of these sugar molecules to proteins remains poorly understood.
Now Chia et al. have discovered 12 molecules that regulate this process, including an enzyme called ERK8 that is found at the Golgi apparatus. ERK8 is shown to prevent the relocation of the sugar-adding enzymes from the Golgi to the endoplasmic reticulum, thereby restricting the production of sugar-coated proteins that help the cancer cells to spread within the body. By identifying 12 potential targets for new therapeutics aimed at preventing the spread of cancer, the work of Chia et al. could ultimately help to improve the chances of patients recovering from certain cancers.
DOI: http://dx.doi.org/10.7554/eLife.01828.002
doi:10.7554/eLife.01828
PMCID: PMC3945522  PMID: 24618899
golgi; glycosylation; cell migration; COP-I; retrograde traffic; endoplasmic reticulum; human; mouse
5.  Mutant enzymes challenge all assumptions 
eLife  2014;3:e02171.
Enzymes called Rab GTPases that carry so-called “activating” mutations may never become activated at all.
doi:10.7554/eLife.02171
PMCID: PMC3919269  PMID: 24520166
Membrane traffic; Rab GTPase; nucleotide exchange factor; Human
6.  Diversity and plasticity in Rab GTPase nucleotide release mechanism has consequences for Rab activation and inactivation 
eLife  2014;3:e01623.
Ras superfamily GTPase activation and inactivation occur by canonical nucleotide exchange and GTP hydrolysis mechanisms. Despite conservation of active-site residues, the Ras-related Rab GTPase activation pathway differs from Ras and between different Rabs. Analysis of DENND1-Rab35, Rabex-Rab5, TRAPP-Rab1 and DrrA-Rab1 suggests Rabs have the potential for activation by distinct GDP-release pathways. Conserved active-site residues in the Rab switch II region stabilising the nucleotide-free form differentiate these pathways. For DENND1-Rab35 and DrrA-Rab1 the Rab active-site glutamine, often mutated to create constitutively active forms, is involved in GEF mediated GDP-release. By contrast, in Rab5 the switch II aspartate is required for Rabex mediated GDP-release. Furthermore, Rab1 switch II glutamine mutants refractory to activation by DrrA can be activated by TRAPP, showing that a single Rab can be activated by more than one mechanistically distinct GDP-release pathway. These findings highlight plasticity in the activation mechanisms of closely related Rab GTPases.
DOI: http://dx.doi.org/10.7554/eLife.01623.001
eLife digest
The 70 or so members of the Rab subfamily of proteins perform a wide range of important tasks inside cells. A Rab protein is always bound to another molecule, which determines whether it is inactive or active. Binding to a molecule called GDP makes the Rab protein inactive, while binding to GTP makes it active. Proteins called guanine nucleotide exchange factors, or GEFs for short, activate the Rab protein by promoting the release of GDP and the binding of GTP. Other proteins—known as GAPs—lead to the inactivation of the Rab protein. Together these proteins form a molecular switch that can be turned on and off.
The Rab subfamily of proteins is part of the large Ras superfamily, and all members of this superfamily are activated and inactivated in a similar way, with the binding and unbinding of GDP and GTP taking place at a structure called the G-domain. The fact that the detailed structure of this domain (at the level of individual amino acids) has been conserved over evolution is often taken as an indication that its mechanism has also been conserved. Langemeyer et al. have now tested this assumption with four different types of GEFs—three from humans and one from the bacteria that cause Listeria—and found that the story is more complicated than expected.
The experiments showed that different amino acids in the active site of the Rab protein are involved when the GEFs mediate the release of the GDP during the activation process. For example, the amino acid glutamine is involved when the Listeria GEF and one of the human GEFs activate the protein, whereas a different amino acid—aspartate—is involved when one of the other human GEFs is responsible for the activation. Using this information, Langemeyer et al. create a human Rab protein that cannot be activated by the GEF from the bacteria that cause Listeria, but can still be activated by its normal human GEF.
By showing that different Rab proteins are activated by different mechanisms, and that a single Rab protein can be activated by more than one mechanism, the work of Langemeyer et al. clearly illustrates the on-going ability of evolution to surprise researchers.
DOI: http://dx.doi.org/10.7554/eLife.01623.002
doi:10.7554/eLife.01623
PMCID: PMC3919270  PMID: 24520163
membrane traffic; Rab GTPase; nucleotide exchange factor; human
7.  Single-molecule analysis reveals self assembly and nanoscale segregation of two distinct cavin subcomplexes on caveolae 
eLife  2014;3:e01434.
In mammalian cells three closely related cavin proteins cooperate with the scaffolding protein caveolin to form membrane invaginations known as caveolae. Here we have developed a novel single-molecule fluorescence approach to directly observe interactions and stoichiometries in protein complexes from cell extracts and from in vitro synthesized components. We show that up to 50 cavins associate on a caveola. However, rather than forming a single coat complex containing the three cavin family members, single-molecule analysis reveals an exquisite specificity of interactions between cavin1, cavin2 and cavin3. Changes in membrane tension can flatten the caveolae, causing the release of the cavin coat and its disassembly into separate cavin1-cavin2 and cavin1-cavin3 subcomplexes. Each of these subcomplexes contain 9 ± 2 cavin molecules and appear to be the building blocks of the caveolar coat. High resolution immunoelectron microscopy suggests a remarkable nanoscale organization of these separate subcomplexes, forming individual striations on the surface of caveolae.
DOI: http://dx.doi.org/10.7554/eLife.01434.001
eLife digest
If you could look closely enough at the surface of some animal cells, especially fat or muscle cells, you would see that they are covered with pocket-like indents called ‘caveolae’. These structures are thought to help the cells communicate with the outside world, but they can also be used by viruses to gain entry into living cells.
Examining these caveolae even closer would reveal that these pockets contain proteins called caveolins that bind to each other—and also to cholesterol and fatty acids—to form a scaffold that help to maintain the shape of the caveolae from inside the cell. Each caveolae in a mammalian cell typically contains over 100 caveolin proteins. Caveolar coat proteins, or cavins for short, are also important building blocks for caveolae: however, we know relatively little about the interactions between caveolins and cavins.
Now, Gambin et al. have used powerful new single-molecule techniques to study these interactions. These experiments looked at the three main types of cavin proteins that associate with caveolae, and by tracking individual protein molecules they showed that cavin1 can interact with either cavin2 or cavin3, but that cavin2 and cavin3 do not interact with each other. Furthermore, cavin2 and cavin3 exist in separate stripes on a caveolae. Gambin et al. also stretched the cell membrane by forcing cells to take in extra water, and showed that this caused the cavin coat to peel away from the caveolae and break down into distinct cavin1-cavin2 and cavin1-cavin3 building blocks.
Faulty versions of caveolins and cavins have both been associated with several diseases in humans, including heart disease and muscle disorders. As such, an improved understanding of the formation and break down of caveolae may prove useful for developing treatments for these conditions.
DOI: http://dx.doi.org/10.7554/eLife.01434.002
doi:10.7554/eLife.01434
PMCID: PMC3903133  PMID: 24473072
caveolae; single-molecule; cell-free protein expression; human
8.  The 5-phosphatase OCRL mediates retrograde transport of the mannose 6-phosphate receptor by regulating a Rac1-cofilin signalling module 
Human Molecular Genetics  2012;21(23):5019-5038.
Mutations in the OCRL gene encoding the phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) 5-phosphatase OCRL cause Lowe syndrome (LS), which is characterized by intellectual disability, cataracts and selective proximal tubulopathy. OCRL localizes membrane-bound compartments and is implicated in intracellular transport. Comprehensive analysis of clathrin-mediated endocytosis in fibroblasts of patients with LS did not reveal any difference in trafficking of epidermal growth factor, low density lipoprotein or transferrin, compared with normal fibroblasts. However, LS fibroblasts displayed reduced mannose 6-phosphate receptor (MPR)-mediated re-uptake of the lysosomal enzyme arylsulfatase B. In addition, endosome-to-trans Golgi network (TGN) transport of MPRs was decreased significantly, leading to higher levels of cell surface MPRs and their enrichment in enlarged, retromer-positive endosomes in OCRL-depleted HeLa cells. In line with the higher steady-state concentration of MPRs in the endosomal compartment in equilibrium with the cell surface, anterograde transport of the lysosomal enzyme, cathepsin D was impaired. Wild-type OCRL counteracted accumulation of MPR in endosomes in an activity-dependent manner, suggesting that PI(4,5)P2 modulates the activity state of proteins regulated by this phosphoinositide. Indeed, we detected an increased amount of the inactive, phosphorylated form of cofilin and lower levels of the active form of PAK3 upon OCRL depletion. Levels of active Rac1 and RhoA were reduced or enhanced, respectively. Overexpression of Rac1 rescued both enhanced levels of phosphorylated cofilin and MPR accumulation in enlarged endosomes. Our data suggest that PI(4,5)P2 dephosphorylation through OCRL regulates a Rac1-cofilin signalling cascade implicated in MPR trafficking from endosomes to the TGN.
doi:10.1093/hmg/dds343
PMCID: PMC3490508  PMID: 22907655
9.  Caveolae internalization repairs wounded cells and muscle fibers 
eLife  2013;2:e00926.
Rapid repair of plasma membrane wounds is critical for cellular survival. Muscle fibers are particularly susceptible to injury, and defective sarcolemma resealing causes muscular dystrophy. Caveolae accumulate in dystrophic muscle fibers and caveolin and cavin mutations cause muscle pathology, but the underlying mechanism is unknown. Here we show that muscle fibers and other cell types repair membrane wounds by a mechanism involving Ca2+-triggered exocytosis of lysosomes, release of acid sphingomyelinase, and rapid lesion removal by caveolar endocytosis. Wounding or exposure to sphingomyelinase triggered endocytosis and intracellular accumulation of caveolar vesicles, which gradually merged into larger compartments. The pore-forming toxin SLO was directly visualized entering cells within caveolar vesicles, and depletion of caveolin inhibited plasma membrane resealing. Our findings directly link lesion removal by caveolar endocytosis to the maintenance of plasma membrane and muscle fiber integrity, providing a mechanistic explanation for the muscle pathology associated with mutations in caveolae proteins.
DOI: http://dx.doi.org/10.7554/eLife.00926.001
eLife digest
Cells must be able to rapidly repair damage to their outer membranes. This is particularly important in the case of muscle cells, which are vulnerable to damage, and the failure of these cells to repair their outer membranes leads to the muscle wastage seen in muscular dystrophy. Researchers do not fully understand how cells repair membrane, but one popular theory is that they use the membranes of specialized vesicles to ‘patch’ areas that have been damaged.
A group of proteins called caveolins have also been implicated in membrane repair but, again, the details have not been worked out. These proteins are best known for their role in the formation of caveolae — small pouches formed by invaginated sections of the plasma membrane. Now, Corrotte et al. have obtained evidence that membrane repair relies not on patching, but on endocytosis (the process by which substances are taken into the cell in small vesicles that ‘pinch’ from the plasma membrane) of these caveolae pouches.
Corrotte et al. treated cells with streptolysin O, a toxin that forms pores in the membrane that cannot be repaired using patches, and found that this led to the formation of small membrane-derived vesicles that looked just like caveolae. Further tests confirmed that these vesicles contained caveolar proteins, and that they removed the toxin from the plasma membrane by endocytosis. Similar effects were seen in response to mechanical damage caused by tiny glass beads. Moreover, blocking the expression of caveolar genes prevented cells from repairing membrane damage.
Based on their findings, Corrotte et al. propose an alternative model for the repair process; namely that cellular damage triggers an influx of calcium ions, which causes vesicles called lysosomes to release chemicals that promote the formation of caveolae. These then remove the damaged area through endocytosis, restoring the integrity of the membrane. The results offer new insights into why mutations in caveolar proteins are associated with muscle disorders, including muscular dystrophy and cardiac dysfunction.
DOI: http://dx.doi.org/10.7554/eLife.00926.002
doi:10.7554/eLife.00926
PMCID: PMC3776555  PMID: 24052812
endocytosis; lysosome; toxin; Human; Mouse
10.  Hopping rim to rim through the Golgi 
eLife  2013;2:e00903.
A novel approach based on tracking the fate of proteins that become ‘stapled’ to the walls of the Golgi yields insights into the long-sought mechanism of transport through this organelle.
doi:10.7554/eLife.00903
PMCID: PMC3679515  PMID: 23795298
Golgi; Traffic; Membrane; Cell biology; Human
11.  Intraflagellar transport drives flagellar surface motility 
eLife  2013;2:e00744.
The assembly and maintenance of all cilia and flagella require intraflagellar transport (IFT) along the axoneme. IFT has been implicated in sensory and motile ciliary functions, but the mechanisms of this relationship remain unclear. Here, we used Chlamydomonas flagellar surface motility (FSM) as a model to test whether IFT provides force for gliding of cells across solid surfaces. We show that IFT trains are coupled to flagellar membrane glycoproteins (FMGs) in a Ca2+-dependent manner. IFT trains transiently pause through surface adhesion of their FMG cargos, and dynein-1b motors pull the cell towards the distal tip of the axoneme. Each train is transported by at least four motors, with only one type of motor active at a time. Our results demonstrate the mechanism of Chlamydomonas gliding motility and suggest that IFT plays a major role in adhesion-induced ciliary signaling pathways.
DOI: http://dx.doi.org/10.7554/eLife.00744.001
eLife digest
Cilia and flagella protrude like bristles from the cell surface. They share the same basic ‘9+2’ axoneme structure, being made up of nine microtubule doublets that surround a central pair of singlet microtubules. Flagella are generally involved in cell propulsion, whereas motile cilia help to move fluids over cell surfaces.
Maintaining cilia and flagella is a challenge for cells, which must find a way to send new proteins all the way along the axoneme to the site of assembly at the flagellar tip. Cells achieve this via a process called intraflagellar transport, in which proteins are carried back and forth by kinesin and dynein motors along the axonemal doublet microtubules. Intraflagellar transport has been proposed to influence other functions of cilia and flagella, including the propulsion of cells over surfaces. However, the details of these interactions are unclear.
Through a combination of biophysical and microscopy approaches, Shih et al. describe the mechanism that the green alga Chalmydomonas uses to power flagellar gliding over surfaces. By tracking single fluorescently tagged molecules, Shih et al. observed that flagellar membrane glycoproteins are carried along the axoneme by the intraflagellar transport machinery. During transport, flagellar membrane glycoproteins transiently adhere to the surface, and dynein motors that were previously engaged in carrying these glycoproteins now transmit force that moves the axonemal microtubules. This process, which is dependent on the concentration of calcium ions in the extracellular environment, generates the force that propels the alga's flagella along the surface.
Gliding motility is thought to have been one of the initial driving forces for the evolution of cilia and flagella. How the intricate mechanism of flagellar beat motility could have evolved has been the subject of much discussion, as it would require the flagellum to have evolved first. In demonstrating that gliding motility is powered by the same intraflagellar transport mechanism that is required for flagellar assembly, Shih et al. provide strong evidence for the evolution of primitive flagella before the evolution of flagellar beating. Furthermore, since algal flagella have essentially the same structure as the cilia of human cells, these findings could ultimately aid in the development of treatments for diseases that result from defects in intraflagellar transport, including polycystic kidney disease and retinal degeneration.
DOI: http://dx.doi.org/10.7554/eLife.00744.002
doi:10.7554/eLife.00744
PMCID: PMC3679542  PMID: 23795295
intraflagellar transport; gliding motility; Chlamydomonas; dynein; kinesin; single molecule; Other
12.  Stapled Golgi cisternae remain in place as cargo passes through the stack 
eLife  2013;2:e00558.
We have designed a membrane ‘staple’, which consists of membrane-anchored repeats of the trans-aggregating FM domain that face the lumen of the secretory pathway. In the presence of the disaggregating drug these proteins transit the secretory pathway. When the drug is removed these proteins form electron-dense plaques which we term staples. Unexpectedly, when initially positioned within the cis-Golgi, staples remained at the cis face of the Golgi even after many hours. By contrast, soluble FM-aggregates transited the Golgi. Staples and soluble aggregates placed in cis-Golgi cisternae therefore have different fates. Whereas the membrane staples are located in the flattened, stacked central regions of the cisternae, the soluble aggregates are in the dilated rims. This suggests that while the cisternae are static on the time scale of protein traffic, the dilated rims are mobile and progress in the cis → trans direction via a mechanism that we term ‘Rim Progression’.
DOI: http://dx.doi.org/10.7554/eLife.00558.001
eLife digest
Most plant and animal cells contain an organelle known as the Golgi apparatus, which consists of a series of four to six stacked cisternae. Almost all the proteins that are secreted from the cell, or targeted to its plasma membrane, transit through the Golgi. This process takes roughly 5–20 min.
Although transport of proteins through the Golgi was first observed more than 50 years ago, it is still unclear exactly how this process occurs. One possibility is that proteins to be packaged move through the cisternae enclosed in vesicles, as if on a conveyor belt. Alternatively, the proteins themselves may remain stationary while the Golgi cisternae move over them.
Now, Lavieu et al. provide evidence that the Golgi shows both mobile and static behaviour depending on the type and size of the cargo being processed. To distinguish between these two mechanisms, they created a new type of protein cargo—which they called a ‘staple’—that became fixed to the walls on each side of the cisternae and could not, therefore, move freely through the Golgi. They compared the processing of this protein to that of a more typical soluble protein cargo, which could move freely through the Golgi stack.
Surprisingly, the Golgi processed these two types of cargo in very different ways. The staples remained embedded in the walls in the center of the cisternae, whereas the conventional soluble cargo was transported past the staples and collected at the edges of the cisternae, which are known as rims. These are wider than the center of the cisternae, and the staples are too narrow to span them. Lavieu et al. suggest that the Golgi cisternae can be divided into two functionally distinct domains: the centers of cisternae, which remain stationary, and the edges or rims, which can move.
In addition to increasing our understanding of how proteins are prepared for transport inside cells, this new mechanism reconciles seemingly conflicting data by revealing that the Golgi can be both mobile and static.
DOI: http://dx.doi.org/10.7554/eLife.00558.002
doi:10.7554/eLife.00558
PMCID: PMC3673335  PMID: 23755362
Golgi; Traffic; Membrane; Human
13.  Cholesterol Accumulation Sequesters Rab9 and Disrupts Late Endosome Function in NPC1-deficient Cells* 
The Journal of biological chemistry  2006;281(26):17890-17899.
Niemann-Pick type C disease is an autosomal recessive disorder that leads to massive accumulation of cholesterol and glycosphingolipids in late endosomes and lysosomes. To understand how cholesterol accumulation influences late endosome function, we investigated the effect of elevated cholesterol on Rab9-dependent export of mannose 6-phosphate receptors from this compartment. Endogenous Rab9 levels were elevated 1.8-fold in Niemann-Pick type C cells relative to wild type cells, and its half-life increased 1.6-fold, suggesting that Rab9 accumulation is caused by impaired protein turnover. Reduced Rab9 degradation was accompaniedby stabilization on endosome membranes, as shown by a reduction in the capacity of Rab9 for guanine nucleotide dissociation inhibitor-mediated extraction from Niemann-Pick type C membranes. Cholesterol appeared to stabilize Rab9 directly, as liposomes loaded with prenylated Rab9 showed decreased extractability with increasing cholesterol content. Rab9 is likely sequestered in an inactive form on Niemann-Pick type C membranes, as cation-dependent man-nose 6-phosphate receptorswere missorted to the lysosome for degradation, a process that was reversed by overexpression of GFP-tagged Rab9. In addition to using primary fibroblasts isolated from Niemann-Pick type C patients, RNA interference was utilized to recapitulate the disease phenotype in cultured cells, greatly facilitating the analysis of cholesterol accumulation and late endosome function. We conclude that cholesterol contributes directly to the sequestration of Rab9 on Niemann-Pick type C cell membranes, which in turn, disrupts mannose 6-phosphate receptor trafficking.
doi:10.1074/jbc.M601679200
PMCID: PMC3650718  PMID: 16644737
14.  Entry at the trans-Face of the Golgi 
The trans-Golgi network (TGN) receives a select set of proteins from the endocytic pathway—about 5% of total plasma membrane glycoproteins (Duncan and Kornfeld 1988). Proteins that are delivered include mannose 6-phosphate receptors (MPRs), TGN46, sortilin, and various toxins that hitchhike a ride backward through the secretory pathway to intoxicate cells after they exit into the cytoplasm from the endoplasmic reticulum (ER). This article will review work on the molecular players that drive protein transport from the endocytic pathway to the TGN. Distinct requirements have revealed multiple routes for retrograde transport; in addition, the existence of multiple, potential coat proteins and/or cargo adaptors imply that multiple vesicular transfers are likely involved. Several comprehensive reviews have appeared recently and should be sought for additional details (Bonifacino and Rojas 2006; Johannes and Popoff 2008).
Proteins such as mannose 6-phosphate receptors and sortilin move to the trans-Golgi network following endocytosis. The retrograde pathways are more complex than expected, requiring numerous adaptor proteins and multiple vesicle transport steps.
doi:10.1101/cshperspect.a005272
PMCID: PMC3039930  PMID: 21421921
15.  A novel GTP-binding protein–adaptor protein complex responsible for export of Vangl2 from the trans Golgi network 
eLife  2013;2:e00160.
Planar cell polarity (PCP) requires the asymmetric sorting of distinct signaling receptors to distal and proximal surfaces of polarized epithelial cells. We have examined the transport of one PCP signaling protein, Vangl2, from the trans Golgi network (TGN) in mammalian cells. Using siRNA knockdown experiments, we find that the GTP-binding protein, Arfrp1, and the clathrin adaptor complex 1 (AP-1) are required for Vangl2 transport from the TGN. In contrast, TGN export of Frizzled 6, which localizes to the opposing epithelial surface from Vangl2, does not depend on Arfrp1 or AP-1. Mutagenesis studies identified a YYXXF sorting signal in the C-terminal cytosolic domain of Vangl2 that is required for Vangl2 traffic and interaction with the μ subunit of AP-1. We propose that Arfrp1 exposes a binding site on AP-1 that recognizes the Vangl2 sorting motif for capture into a transport vesicle destined for the proximal surface of a polarized epithelial cell.
DOI: http://dx.doi.org/10.7554/eLife.00160.001
eLife digest
Most cells in multicellular organisms possess a property known as polarity that is reflected, in part, in the organization of the cell surface into distinct domains. One well-known axis in epithelial cells, such as those in the skin, divides the cell into an apical domain, which faces out, and a basal domain, which faces the underlying tissue. These cells rely on the distribution of structural components inside the cell, or within the cell membrane, to tell the difference between these two directions. Epithelial cells also possess a second type of polarity, planar cell polarity, that ensures that cells adjacent to each other in the plane parallel to the skin tissue are oriented correctly with respect to each other during development. This ensures, in turn, that hairs, scales, feathers and so on are all aligned.
All eukaryotic cells sort and process proteins within an organelle called the Golgi apparatus, and proteins that are required at a specific destination within the cell, such as the cell surface membrane, carry specific molecular sorting signals that act as address labels to convey the protein into and within the secretory pathway. As one of these proteins moves through the Golgi apparatus, its sorting signals are recognized by coat proteins, such as clathrin, that subsequently form a vesicle around it. The assembly of this vesicle is initiated by an enzyme from the Arf family, but the enzyme must first undergo a conformational change (by exchanging a molecule of GDP for one of GTP) before formation can begin. The resulting vesicle can then be sent on its way to the address indicated by its Golgi-to-cell-surface sorting signal. These sorting signals also help to establish planar cell polarity in cells by ensuring that proteins called signaling receptors are distributed asymmetrically within the cell membrane.
Guo et al. have now examined the mechanism behind the asymmetric sorting of two proteins that are involved in planar cell polarity: Vangl2 and Frizzled 6. In an effort to understand why these proteins are localized to opposite surfaces of epithelial cells, Guo et al. used genetic techniques to reduce the expression of Golgi-localized Arf proteins in epithelial cell cultures. They found that knockdown of a protein called Arfrp1 caused Vangl2 to accumulate in the last station of the Golgi complex instead of being transported to the cell surface membrane. Then, using a technique called affinity chromatography, they demonstrated that a coat protein called the clathrin adaptor complex (AP-1) had to be present for the formation of vesicles around Vangl2. Moreover, disrupting AP-1 and Arfrp1 did not prevent Frizzled 6 being transported to the cell surface membrane. This suggests that cells use several distinct adaptor proteins and coat complexes to ensure that proteins from the Golgi apparatus go to specific locations on the cell surface and, thus, help to establish planar cell polarity.
DOI: http://dx.doi.org/10.7554/eLife.00160.002
doi:10.7554/eLife.00160
PMCID: PMC3539332  PMID: 23326640
TGN sorting; Vesicle coat proteins; Arf proteins; Human
16.  GCC185 plays independent roles in Golgi structure maintenance and AP-1–mediated vesicle tethering 
The Journal of Cell Biology  2011;194(5):779-787.
Two distinct domains of GCC185 function in maintaining Golgi structure or in binding to AP-1 to tether retrograde transport vesicles en route to the Golgi.
GCC185 is a long coiled-coil protein localized to the trans-Golgi network (TGN) that functions in maintaining Golgi structure and tethering mannose 6-phosphate receptor (MPR)–containing transport vesicles en route to the Golgi. We report the identification of two distinct domains of GCC185 needed either for Golgi structure maintenance or transport vesicle tethering, demonstrating the independence of these two functions. The domain needed for vesicle tethering binds to the clathrin adaptor AP-1, and cells depleted of GCC185 accumulate MPRs in transport vesicles that are AP-1 decorated. This study supports a previously proposed role of AP-1 in retrograde transport of MPRs from late endosomes to the Golgi and indicates that docking may involve the interaction of vesicle-associated AP-1 protein with the TGN-associated tethering protein GCC185.
doi:10.1083/jcb.201104019
PMCID: PMC3171126  PMID: 21875948
17.  Recent advances in understanding Golgi biogenesis 
The Golgi complex is a central processing station for proteins traversing the secretory pathway, yet we are still learning how this compartment is constructed and how cargo moves through it. Recent experiments suggest a key role for Ras-like Rab GTPases and provide important new ideas for how the Golgi may function.
doi:10.3410/B2-32
PMCID: PMC2897732  PMID: 20625450
18.  Multiple routes of protein transport from endosomes to the trans Golgi network 
FEBS letters  2009;583(23):3811-3816.
Proteins use multiple routes for transport from endosomes to the Golgi complex. Shiga and cholera toxins and TGN38/46 are routed from early and recycling endosomes, while mannose 6-phosphate receptors are routed from late endosomes. The identification of distinct molecular requirements for each of these pathways makes it clear that mammalian cells have evolved more complex targeting mechanisms and routes than previously anticipated.
doi:10.1016/j.febslet.2009.10.075
PMCID: PMC2787657  PMID: 19879268
endosome; Golgi; Rab GTPase; mannose 6-phosphate receptors; Shiga and cholera toxins
19.  Unconventional secretion by autophagosome exocytosis 
The Journal of Cell Biology  2010;188(4):451-452.
In this issue, Duran et al. (2010. J. Cell Biol. doi: 10.1083/jcb.200911154) and Manjithaya et al. (2010. J. Cell Biol. doi: 10.1083/jcb.200911149) use yeast genetics to reveal a role for autophagosome intermediates in the unconventional secretion of an acyl coenzyme A (CoA)–binding protein that lacks an endoplasmic reticulum signal sequence. Medium-chain acyl CoAs are also required and may be important for substrate routing to this pathway.
doi:10.1083/jcb.201001121
PMCID: PMC2828920  PMID: 20156968
20.  Association of β-1,3-N-acetylglucosaminyltransferase 1 and β-1,4-galactosyltransferase 1, trans-Golgi enzymes involved in coupled poly-N-acetyllactosamine synthesis 
Glycobiology  2009;19(6):655-664.
Poly-N-acetyllactosamine (polyLacNAc) is a linear carbohydrate polymer composed of alternating N-acetylglucosamine and galactose residues involved in cellular functions ranging from differentiation to metastasis. PolyLacNAc also serves as a scaffold on which other oligosaccharides such as sialyl Lewis X are displayed. The polymerization of the alternating N-acetylglucosamine and galactose residues is catalyzed by the successive action of UDP-GlcNAc:βGal β-1,3-N-acetylglucosaminyltransferase 1 (B3GNT1) and UDP-Gal:βGlcNAc β-1,4-galactosyltransferase, polypeptide 1 (B4GALT1), respectively. The functional association between these two glycosyltransferases led us to investigate whether the enzymes also associate physically. We show that B3GNT1 and B4GALT1 colocalize by immunofluorescence microscopy, interact by coimmunoprecipitation, and affect each other's subcellular localization when one of the two proteins is artificially retained in the endoplasmic reticulum. These results demonstrate that B3GNT1 and B4GALT1 physically associate in vitro and in cultured cells, providing insight into possible mechanisms for regulation of polyLacNAc production.
doi:10.1093/glycob/cwp035
PMCID: PMC2682609  PMID: 19261593
endoplasmic reticulum; enzyme complexes; glycosyltransferase; Golgi complex; poly-N-acetyllactosamine
21.  RhoBTB3: A Rho GTPase-family ATPase required for endosome to Golgi transport 
Cell  2009;137(5):938-948.
Summary
Rho GTPases are key regulators of the actin-based cytoskeleton; Rab GTPases are key regulators of membrane traffic. We report here that the atypical Rho GTPase family member, RhoBTB3, binds directly to Rab9 GTPase, and functions with Rab9 in protein transport from endosomes to the trans Golgi network. Gene replacement experiments show that RhoBTB3 function in cultured cells requires both RhoBTB3’s N-terminal, Rho-related domain, and C-terminal sequences that are important for Rab9 interaction.9 Biochemical analysis reveals that RhoBTB3 binds and hydrolyzes ATP rather than GTP. Rab9 binding opens the auto-inhibited RhoBTB3 protein to permit maximal ATP hydroysis. Because RhoBTB3 interacts with TIP47 on membranes, we propose that it may function to release this cargo selection protein from vesicles to permit their efficient docking and fusion at the Golgi.
doi:10.1016/j.cell.2009.03.043
PMCID: PMC2801561  PMID: 19490898
22.  Journeys through the Golgi—taking stock in a new era 
The Journal of Cell Biology  2009;187(4):449-453.
The Golgi apparatus is essential for protein sorting and transport. Many researchers have long been fascinated with the form and function of this organelle. Yet, despite decades of scrutiny, the mechanisms by which proteins are transported across the Golgi remain controversial. At a recent meeting, many prominent Golgi researchers assembled to critically evaluate the core issues in the field. This report presents the outcome of their discussions and highlights the key open questions that will help guide the field into a new era.
doi:10.1083/jcb.200909011
PMCID: PMC2779233  PMID: 19948493
23.  Recent advances in understanding Golgi biogenesis 
The Golgi complex is a central processing station for proteins traversing the secretory pathway, yet we are still learning how this compartment is constructed and how cargo moves through it. Recent experiments suggest a key role for Ras-like Rab GTPases and provide important new ideas for how the Golgi may function.
doi:10.3410/B2-32
PMCID: PMC2897732  PMID: 20625450
24.  Multiple Rab GTPase Binding Sites in GCC185 Suggest a Model for Vesicle Tethering at the Trans-Golgi 
Molecular Biology of the Cell  2009;20(1):209-217.
GCC185, a trans-Golgi network-localized protein predicted to assume a long, coiled-coil structure, is required for Rab9-dependent recycling of mannose 6-phosphate receptors (MPRs) to the Golgi and for microtubule nucleation at the Golgi via CLASP proteins. GCC185 localizes to the Golgi by cooperative interaction with Rab6 and Arl1 GTPases at adjacent sites near its C terminus. We show here by yeast two-hybrid and direct biochemical tests that GCC185 contains at least four additional binding sites for as many as 14 different Rab GTPases across its entire length. A central coiled-coil domain contains a specific Rab9 binding site, and functional assays indicate that this domain is important for MPR recycling to the Golgi complex. N-Terminal coiled-coils are also required for GCC185 function as determined by plasmid rescue after GCC185 depletion by using small interfering RNA in cultured cells. Golgi-Rab binding sites may permit GCC185 to contribute to stacking and lateral interactions of Golgi cisternae as well as help it function as a vesicle tether.
doi:10.1091/mbc.E08-07-0740
PMCID: PMC2613123  PMID: 18946081
25.  Hrs and SNX3 Functions in Sorting and Membrane Invagination within Multivesicular Bodies  
PLoS Biology  2008;6(9):e214.
After internalization, ubiquitinated signaling receptors are delivered to early endosomes. There, they are sorted and incorporated into the intralumenal invaginations of nascent multivesicular bodies, which function as transport intermediates to late endosomes. Receptor sorting is achieved by Hrs—an adaptor-like protein that binds membrane PtdIns3P via a FYVE motif—and then by ESCRT complexes, which presumably also mediate the invagination process. Eventually, intralumenal vesicles are delivered to lysosomes, leading to the notion that EGF receptor sorting into multivesicular bodies mediates lysosomal targeting. Here, we report that Hrs is essential for lysosomal targeting but dispensable for multivesicular body biogenesis and transport to late endosomes. By contrast, we find that the PtdIns3P-binding protein SNX3 is required for multivesicular body formation, but not for EGF receptor degradation. PtdIns3P thus controls the complementary functions of Hrs and SNX3 in sorting and multivesicular body biogenesis.
Author Summary
The cell's genetic program is modulated by extracellular signals that activate cell surface receptors and, in turn, intracellular effectors, to regulate transcription. For cells to function normally, these signals must be turned off to avoid permanent activation—a situation often associated with cancer. For many receptors, signaling is repressed, or down-regulated, in a process that first internalizes and then degrades the receptors. After receptors are removed from the cell surface into structures called early endosomes, they are selectively incorporated within vesicles that form inside the endosome. During this process, endosomal membranes are pulled away from the cytoplasm towards the endosome lumen, against the flow of intracellular membrane traffic, eventually resulting in the formation of a “multivesicular body” (vesicles within vesicles). The common view is that these intralumenal vesicles are then delivered to lysosomes, where they are degraded along with their receptor cargo. We have investigated the mechanisms responsible for the biogenesis of intralumenal vesicles in multivesicular bodies. We find that the small protein SNX3, which binds the signaling lipid phosphatidyl inositol-3-phosphate, is necessary for the formation of intralumenal vesicles, but is not involved in the degradation of the cell surface receptor for EGF. Conversely, we find that Hrs, which also binds phosphatidyl inositol-3-phosphate and mediates receptor sorting into intralumenal vesicles, is essential for lysosomal targeting but dispensable for multivesicular body biogenesis. Phosphatidyl inositol-3-phosphate thus controls the complementary functions of Hrs and SNX3 in the sorting of signaling receptors and multivesicular body biogenesis.
SNX3 plays a direct role in the formation of intralumenal vesicles of multivesicular bodies (MVBs) but is not involved in EGF receptor degradation, whereas Hrs is essential for lysosomal targeting but dispensable for MVB biogenesis. Hence, intralumenal vesicle formation in MVB biogenesis can be uncoupled from lysosomal targeting.
doi:10.1371/journal.pbio.0060214
PMCID: PMC2528051  PMID: 18767904

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