Most kinesins transport cargoes bound to their C-termini and use N-terminal motor domains to move along microtubules. We report here a novel function for KIF1C: it transports Rab6A-vesicles and can influence Golgi complex organization. These activities correlate with KIF1C's capacity to bind the Golgi protein Rab6A directly, both via its motor domain and C-terminus. Rab6A binding to the motor domain inhibits microtubule interaction in vitro and in cells, decreasing the amount of motile KIF1C. KIF1C depletion slows protein delivery to the cell surface, interferes with vesicle motility, and triggers Golgi fragmentation. KIF1C can protect Golgi membranes from fragmentation in cells lacking an intact microtubule network. Rescue of fragmentation requires sequences that enable KIF1C to bind Rab6A at both ends, but not KIF1C motor function. Rab6A binding to KIF1C's motor domain represents an entirely new mode of regulation for a kinesin motor, and likely has important consequences for KIF1C's cellular functions.
Within our cells there are many compartments that play important roles. Small bubble-like packages called vesicles carry proteins and other molecules between these compartments. These vesicles can be driven around cells by a family of motor proteins called kinesins, which move along a network of filaments called microtubules.
Kinesin proteins have two sections known as the N-terminus and the C-terminus. In most cases, the N-terminus contains the motor that binds to and walks along microtubules, while the C-terminus binds to vesicles or other cell compartments. Attached to the compartments are members of another family of proteins called the Rab GTPases. These proteins help the kinesins bind to a compartment, but it was not clear if, or how, these proteins control the activity of the kinesins.
Here, Lee et al. studied a kinesin called KIF1C. The experiments show that this kinesin can move vesicles that contain a Rab-GTPase called Rab6A along microtubules. Unexpectedly, Rab6A controls the activity of KIF1C by directly interacting with the motor as well as the C-terminus. Loss of the kinesin from the cell slows down the delivery of cargo carried in vesicles to the surface of the cell.
The experiments also show that KIF1C is involved in organizing another compartment within cells called the Golgi. This role relies on Rab6A binding to both the N-terminus and C-terminus of the kinesin, but does not require the kinesin to act as a motor. Lee et al.'s findings reveal a new way in which the activity of kinesins can be controlled. Future challenges will be to find out if other kinesins are also controlled in this way and discover when and where the Rab GTPases bind motor domains in cells.
Golgi complex; kinesin; Rab GTPase; KIF1C; microtubule motor; none
The Golgi is decorated with coiled-coil proteins that may extend long distances to help vesicles find their targets. GCC185 is a trans Golgi-associated protein that captures vesicles inbound from late endosomes. Although predicted to be relatively rigid and highly extended, we show that flexibility in a central region is required for GCC185’s ability to function in a vesicle tethering cycle. Proximity ligation experiments show that that GCC185’s N-and C-termini are within <40 nm of each other on the Golgi. In physiological buffers without fixatives, atomic force microscopy reveals that GCC185 is shorter than predicted, and its flexibility is due to a central bubble that represents local unwinding of specific sequences. Moreover, 85% of the N-termini are splayed, and the splayed N-terminus can capture transport vesicles in vitro. These unexpected features support a model in which GCC185 collapses onto the Golgi surface, perhaps by binding to Rab GTPases, to mediate vesicle tethering.
Some cells release molecules, such as hormones and neurotransmitters, to signal to other cells and influence how they work. As part of the release process, these molecules are packaged into small, balloon-like structures called vesicles. Such vesicles move around within cells and are able to find the right place to release their contents to the outside.
A cellular compartment called the Golgi complex helps to prepare proteins for release from the cell. Vesicles can bind to tethering proteins on the surface of the Golgi, but it was not clear how these proteins are able to capture the correct kind of vesicle. The prediction was that the proteins are rigid, shaped like pipe cleaners that stick out from the Golgi as a meshwork that traps vesicles.
Cheung et al. isolated a specific Golgi tethering protein (called GCC185) from cultured human cells and used a technique called atomic force microscopy to visualize its structure. This revealed that this protein is not rod-like; it is instead rather floppy, and has two arms at one end that may ‘hug’ the incoming vesicle. Cheung et al. showed that this protein needs its middle portion to be floppy to work correctly. This changes the way we think about how vesicles are able to find their corresponding targets on different compartments inside cells.
Further experiments are now needed to answer a number of questions. What does the tether look like when actually bound to a vesicle? What happens after the vesicle binds – how does the tether let go? What other components are needed for vesicle capture and release?
coiled-coil; membrane traffic; vesicle tethering; Golgi complex; Human
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.
Enzymes called Rab GTPases that carry so-called “activating” mutations may never become activated at all.
Membrane traffic; Rab GTPase; nucleotide exchange factor; Human
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.
Golgi; Traffic; Membrane; Cell biology; Human
The 2013 Nobel Prize in Physiology or Medicine has been awarded to James Rothman, Randy Schekman, and Thomas Südhof “for their discoveries of machinery regulating vesicle traffic, a major transport system in our cells”. I present a personal view of the membrane trafficking field, highlighting the contributions of these three Nobel laureates in a historical context.
Rab GTPases are master regulators of membrane traffic. By binding to distinct sets of effector proteins, Rabs catalyse the formation of function-specifying membrane microdomains. They are delivered to membranes by a protein named GDI (guanine-nucleotide-dissociation inhibitor) and are stabilized there after nucleotide exchange by effector binding. In the present mini-review, I discuss what we know about how Rab GTPases are delivered to the correct membrane-bound compartments and how Rab GTPase cascades order Rabs within the secretory and endocytic pathways. Finally, I describe how Rab cascades may establish the distinct compartments of the Golgi complex to permit ordered processing, sorting and secretion of secretory cargoes.
guanine-nucleotide-dissociation inhibitor displacement factor (GDF); guanine-nucleotide-dissociation inhibitor (GDI); Golgi complex; GTPase-activating protein (GAP); guanine-nucleotide-exchange factor (GEF); Rab GTPase
Mammalian cells encode a diverse set of Rab GTPases and their corresponding regulators. In vitro biochemical screening has proven invaluable in assigning particular Rabs as substrates for their cognate GTPase-activating proteins. However, in vitro activity does not always reflect substrate specificity in cells. This method describes a functional test of GAP activity in cells or extracts that takes into account the presence of other factors or conditions that might change observed in vitro specificity.
GTPase-activating protein; Rab GTPase; Effector protein
Transport vesicle tethers are proteins that link partner membranes together to permit subsequent SNARE protein pairing and fusion. Despite the identification of a relatively large number of tethering proteins, little is known about the precise mechanisms by which they act. Biochemical isolation of tethers permits direct analysis of their physical characteristics and molecular interactions. Here, we describe the expression and purification of GCC185, a trans-Golgi-localized, 190-kDa coiled-coil tethering protein. In addition, we present a gene rescue approach to analyze the function of this tether after its depletion from cells using siRNA.
Golgi complex; Membrane traffic; Tethering factor; Secretion; Protein purification
Sorting nexin proteins (SNXs) and the cargo-selective retromer complex play key roles in receptor recycling from endosomes to the cell surface. A global proteomics analysis reveals a collection of cell surface proteins that rely on SNX27 and the retromer complex for their cell surface localization at steady state.
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.
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.
Mannose 6-phosphate receptors (MPRs) are transported from endosomes to the Golgi after delivering lysosomal enzymes to the endocytic pathway. This process requires Rab9 guanosine triphosphatase (GTPase) and the putative tether GCC185. We show in human cells that a soluble NSF attachment protein receptor (SNARE) complex comprised of syntaxin 10 (STX10), STX16, Vti1a, and VAMP3 is required for this MPR transport but not for the STX6-dependent transport of TGN46 or cholera toxin from early endosomes to the Golgi. Depletion of STX10 leads to MPR missorting and hypersecretion of hexosaminidase. Mouse and rat cells lack STX10 and, thus, must use a different target membrane SNARE for this process. GCC185 binds directly to STX16 and is competed by Rab6. These data support a model in which the GCC185 tether helps Rab9-bearing transport vesicles deliver their cargo to the trans-Golgi and suggest that Rab GTPases can regulate SNARE–tether interactions. Importantly, our data provide a clear molecular distinction between the transport of MPRs and TGN46 to the trans-Golgi.
Mulitmeric cullin-RING ubiquitin ligases (CRLs) represent the largest class of ubiquitin ligases in eukaryotes. However, most CRL ubiquitylation pathways remain uncharacterized. CRLs control a myriad of functions by catalyzing mono- or poly-ubiquitylation of target proteins. Recently, novel CRLs have been identified along the secretory pathway where they modify substrates involved in diverse cellular processes such as vesicle coat assembly and cell cycle progression. This review discusses our current understanding of CRL ubiquitylation within the secretory pathway, with special emphasis on the emerging role of the Golgi as a ubiquitylation platform. CRLs are also implicated in endosome function, where their specific roles are less well understood.
Cullin proteins; ubiquitylation; Golgi complex; cullin-RING ligases; post-translational modification
Mannose 6-phosphate receptors (MPRs) deliver newly synthesized lysosomal enzymes to endosomes and then recycle to the Golgi. MPR recycling requires Rab9 GTPase; Rab9 recruits the cytosolic adaptor TIP47 and enhances its ability to bind to MPR cytoplasmic domains during transport vesicle formation. Rab9-bearing vesicles then fuse with the trans-Golgi network (TGN) in living cells, but nothing is known about how these vesicles identify and dock with their target. We show here that GCC185, a member of the Golgin family of putative tethering proteins, is a Rab9 effector that is required for MPR recycling from endosomes to the TGN in living cells, and in vitro. GCC185 does not rely on Rab9 for its TGN localization; depletion of GCC185 slightly alters the Golgi ribbon but does not interfere with Golgi function. Loss of GCC185 triggers enhanced degradation of mannose 6-phosphate receptors and enhanced secretion of hexosaminidase. These data assign a specific pathway to an interesting, TGN-localized protein and suggest that GCC185 may participate in the docking of late endosome-derived, Rab9-bearing transport vesicles at the TGN.
Mannose 6-phosphate receptors (MPRs) are transported from endosomes to the trans-Golgi via a transport process that requires the Rab9 GTPase and the cargo adaptor TIP47. We have generated green fluorescent protein variants of Rab9 and determined their localization in cultured cells. Rab9 is localized primarily in late endosomes and is readily distinguished from the trans-Golgi marker galactosyltransferase. Coexpression of fluorescent Rab9 and Rab7 revealed that these two late endosome Rabs occupy distinct domains within late endosome membranes. Cation-independent mannose 6-phosphate receptors are enriched in the Rab9 domain relative to the Rab7 domain. TIP47 is likely to be present in this domain because it colocalizes with the receptors in fixed cells, and a TIP47 mutant disrupted endosome morphology and sequestered MPRs intracellularly. Rab9 is present on endosomes that display bidirectional microtubule-dependent motility. Rab9-positive transport vesicles fuse with the trans-Golgi network as followed by video microscopy of live cells. These data provide the first indication that Rab9-mediated endosome to trans-Golgi transport can use a vesicle (rather than a tubular) intermediate. Our data suggest that Rab9 remains vesicle associated until docking with the Golgi complex and is rapidly removed concomitant with or just after membrane fusion.
endosome; Rab9; Golgi complex; Rab7; TIP47
In this issue, Short et al. report the discovery of a protein named Golgin-45 that is located on the surface of the middle (or medial) cisternae of the Golgi complex. Depletion of this protein disrupts the Golgi complex and leads to the return of a resident, lumenal, medial Golgi enzyme to the endoplasmic reticulum. These findings suggest that Golgin-45 serves as a linchpin for the maintenance of Golgi complex structure, and offer hints as to the mechanisms by which the polarized Golgi complex is constructed.
Membrane trafficking involves the collection of cargo into nascent transport vesicles that bud off from a donor compartment, translocate along cytoskeletal tracks, and then dock and fuse with their target membranes. Docking and fusion involve initial interaction at a distance (tethering), followed by a closer interaction that leads to pairing of vesicle SNARE proteins (v-SNAREs) with target membrane SNAREs (t-SNAREs), thereby catalyzing vesicle fusion. When tethering cannot take place, transport vesicles accumulate in the cytoplasm. Tethering is generally carried out by two broad classes of molecules: extended, coiled-coil proteins such as the so-called Golgin proteins, or multi-subunit complexes such as the Exocyst, COG or Dsl complexes. This review will focus on the most recent advances in terms of our understanding of the mechanism by which tethers carry out their roles, and new structural insights into tethering complex transactions.
Membrane traffic; vesicle tethering; Rab GTPase; Golgi complex; endosomes
A fundamental question in cell biology is how cells determine membrane compartment identity and the directionality with which cargoes pass through the secretory and endocytic pathways. The discovery of so-called “Rab cascades” provides a satisfying molecular mechanism that helps to resolve this paradox. One Rab GTPase has the ability to template the localization of the subsequent acting Rab GTPase along a given transport pathway. Thus, in addition to determining compartment identity and functionality, Rab GTPases are likely able to order the events of membrane trafficking. This review will highlight recent advances in our understanding of Rabs and Rab cascades.
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.
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.
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.
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.
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.
endosome; Golgi; Rab GTPase; mannose 6-phosphate receptors; Shiga and cholera toxins
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.