Insulin stimulates glucose transport by delivering GLUT4 from a specialized storage compartment to the plasma membrane. Subcellular fractionation and an in vitro assay for GLUT4-storage vesicle formation show that the Sec1/Munc18 protein mVps45 is required to correctly sort GLUT4 into this compartment.
Insulin stimulates glucose transport in fat and muscle cells by regulating delivery of the facilitative glucose transporter, glucose transporter isoform 4 (GLUT4), to the plasma membrane. In the absence of insulin, GLUT4 is sequestered away from the general recycling endosomal pathway into specialized vesicles, referred to as GLUT4-storage vesicles. Understanding the sorting of GLUT4 into this store is a major challenge. Here we examine the role of the Sec1/Munc18 protein mVps45 in GLUT4 trafficking. We show that mVps45 is up-regulated upon differentiation of 3T3-L1 fibroblasts into adipocytes and is expressed at stoichiometric levels with its cognate target–soluble N-ethylmaleimide–sensitive factor attachment protein receptor, syntaxin 16. Depletion of mVps45 in 3T3-L1 adipocytes results in decreased GLUT4 levels and impaired insulin-stimulated glucose transport. Using subcellular fractionation and an in vitro assay for GLUT4-storage vesicle formation, we show that mVps45 is required to correctly traffic GLUT4 into this compartment. Collectively our data reveal a crucial role for mVps45 in the delivery of GLUT4 into its specialized, insulin-regulated compartment.
Reconstitution of GLUT4 vesicle fusion in a defined fusion system shows that the C2-domain factor Doc2b activates the SNARE-dependent fusion reaction by a calcium- and membrane bending–dependent mechanism. Of importance, certain features of Doc2b function appear to be distinct from how synaptotagmin-1 promotes synaptic release.
The glucose transporter GLUT4 plays a central role in maintaining body glucose homeostasis. On insulin stimulation, GLUT4-containing vesicles fuse with the plasma membrane, relocating GLUT4 from intracellular reservoirs to the cell surface to uptake excess blood glucose. The GLUT4 vesicle fusion reaction requires soluble N-ethylmaleimide–sensitive factor attachment protein receptors (SNAREs) as the core fusion engine and a group of regulatory proteins. In particular, the soluble C2-domain factor Doc2b plays a key role in GLUT4 vesicle fusion, but its molecular mechanism has been unclear. Here we reconstituted the SNARE-dependent GLUT4 vesicle fusion in a defined proteoliposome fusion system. We observed that Doc2b binds to GLUT4 exocytic SNAREs and potently accelerates the fusion kinetics in the presence of Ca2+. The stimulatory activity of Doc2b requires intact Ca2+-binding sites on both the C2A and C2B domains. Using electron microscopy, we observed that Doc2b strongly bends the membrane bilayer, and this membrane-bending activity is essential to the stimulatory function of Doc2b in fusion. These results demonstrate that Doc2b promotes GLUT4 exocytosis by accelerating the SNARE-dependent fusion reaction by a Ca2+- and membrane bending–dependent mechanism. Of importance, certain features of Doc2b function appear to be distinct from how synaptotagmin-1 promotes synaptic neurotransmitter release, suggesting that exocytic Ca2+ sensors may possess divergent mechanisms in regulating vesicle fusion.
Highly motile fungal early endosomes can be easily distinguished from more static late endosomes and vacuoles, a feature that is exploited to study endosomal maturation. RabA/RabB early endosomes mature into RabSRab7 late endosomes as they move away from the tip where endocytosis predominates, augmenting their size, with concomitant loss of motility.
We exploit the ease with which highly motile early endosomes are distinguished from static late endosomes in order to study Aspergillus nidulans endosomal traffic. RabSRab7 mediates homotypic fusion of late endosomes/vacuoles in a homotypic fusion- and vacuole protein sorting/Vps41–dependent manner. Progression across the endocytic pathway involves endosomal maturation because the end products of the pathway in the absence of RabSRab7 are minivacuoles that are competent in multivesicular body sorting and cargo degradation but retain early endosomal features, such as the ability to undergo long-distance movement and propensity to accumulate in the tip region if dynein function is impaired. Without RabSRab7, early endosomal Rab5s—RabA and RabB—reach minivacuoles, in agreement with the view that Rab7 homologues facilitate the release of Rab5 homologues from endosomes. RabSRab7 is recruited to membranes already at the stage of late endosomes still lacking vacuolar morphology, but the transition between early and late endosomes is sharp, as only in a minor proportion of examples are RabA/RabB and RabSRab7 detectable in the same—frequently the less motile—structures. This early-to-late endosome/vacuole transition is coupled to dynein-dependent movement away from the tip, resembling the periphery-to-center traffic of endosomes accompanying mammalian cell endosomal maturation. Genetic studies establish that endosomal maturation is essential, whereas homotypic vacuolar fusion is not.
During oogenesis in Drosophila, the phagocytic engulfment protein Ced-6 recognizes the atypical endocytic sorting signal within the vitellogenin receptor Yolkless. Because Ced-6 displays all of the features of an authentic clathrin adaptor, an unrecognized clathrin dependence for Ced-6/Gulp operation during phagocytosis is possible.
Clathrin-mediated endocytosis and phagocytosis are both selective surface internalization processes but have little known mechanistic similarity or interdependence. Here we show that the phosphotyrosine-binding (PTB) domain protein Ced-6, a well-established phagocytosis component that operates as a transducer of so-called “eat-me” signals during engulfment of apoptotic cells and microorganisms, is expressed in the female Drosophila germline and that Ced-6 expression correlates with ovarian follicle development. Ced-6 exhibits all the known biochemical properties of a clathrin-associated sorting protein, yet ced-6–null flies are semifertile despite massive accumulation of soluble yolk precursors in the hemolymph. This is because redundant sorting signals within the cytosolic domain of the Drosophila vitellogenin receptor Yolkless, a low density lipoprotein receptor superfamily member, occur; a functional atypical dileucine signal binds to the endocytic AP-2 clathrin adaptor directly. Nonetheless, the Ced-6 PTB domain specifically recognizes the noncanonical Yolkless FXNPXA sorting sequence and in HeLa cells promotes the rapid, clathrin-dependent uptake of a Yolkless chimera lacking the distal dileucine signal. Ced-6 thus operates in vivo as a clathrin adaptor. Because the human Ced-6 orthologue GULP similarly binds to clathrin machinery, localizes to cell surface clathrin-coated structures, and is enriched in placental clathrin-coated vesicles, new possibilities for Ced-6/Gulp operation during phagocytosis must be considered.
The Sec6 subunit of the multisubunit exocyst tethering complex interacts with the Sec1/Munc18 protein Sec1 and with the t-SNARE Sec9. Assembly of the exocyst upon vesicle arrival at sites of secretion is proposed to release Sec9 for SNARE complex assembly and to recruit Sec1 for interaction with SNARE complexes to facilitate fusion.
Trafficking of protein and lipid cargo through the secretory pathway in eukaryotic cells is mediated by membrane-bound vesicles. Secretory vesicle targeting and fusion require a conserved multisubunit protein complex termed the exocyst, which has been implicated in specific tethering of vesicles to sites of polarized exocytosis. The exocyst is directly involved in regulating soluble N-ethylmaleimide–sensitive factor (NSF) attachment protein receptor (SNARE) complexes and membrane fusion through interactions between the Sec6 subunit and the plasma membrane SNARE protein Sec9. Here we show another facet of Sec6 function—it directly binds Sec1, another SNARE regulator, but of the Sec1/Munc18 family. The Sec6–Sec1 interaction is exclusive of Sec6–Sec9 but compatible with Sec6–exocyst assembly. In contrast, the Sec6–exocyst interaction is incompatible with Sec6–Sec9. Therefore, upon vesicle arrival, Sec6 is proposed to release Sec9 in favor of Sec6–exocyst assembly and to simultaneously recruit Sec1 to sites of secretion for coordinated SNARE complex formation and membrane fusion.
This study investigates the hypothesis that trafficking of membrane proteins occurs at plasma membrane (PM) domains adjacent to underlying cortical endoplasmic reticulum (cER). The authors observe exocytosis of transferrin receptor and vesicular stomatitis virus G-protein to occur preferentially (>80%) at cER-enriched PM domains. They also report a preferential (>80%) localization of clathrin-coated pits at these domains.
In mammalian cells, the cortical endoplasmic reticulum (cER) is a network of tubules and cisterns that lie in close apposition to the plasma membrane (PM). We provide evidence that PM domains enriched in underlying cER function as trafficking hubs for insertion and removal of PM proteins in HEK 293 cells. By simultaneously visualizing cER and various transmembrane protein cargoes with total internal reflectance fluorescence microscopy, we demonstrate that the majority of exocytotic delivery events for a recycled membrane protein or for a membrane protein being delivered to the PM for the first time occur at regions enriched in cER. Likewise, we observed recurring clathrin clusters and functional endocytosis of PM proteins preferentially at the cER-enriched regions. Thus the cER network serves to organize the molecular machinery for both insertion and removal of cell surface proteins, highlighting a novel role for these unique cellular microdomains in membrane trafficking.
The meiosis II outer plaque (MOP) acts a vesicle tethering complex that is a site for de novo membrane formation. Novel mutants in a MOP protein reveal that interaction of vesicles with the MOP surface is required to recruit a second tethering complex, the exocyst, to the vesicles, suggesting a mechanism by which the MOP promotes vesicle fusion.
During meiosis II in Saccharomyces cerevisiae, the cytoplasmic face of the spindle pole body, referred to as the meiosis II outer plaque (MOP), is modified in both composition and structure to become the initiation site for de novo formation of a membrane called the prospore membrane. The MOP serves as a docking complex for precursor vesicles that are targeted to its surface. Using fluorescence resonance energy transfer analysis, the orientation of coiled-coil proteins within the MOP has been determined. The N-termini of two proteins, Mpc54p and Spo21p, were oriented toward the outer surface of the structure. Mutations in the N-terminus of Mpc54p resulted in a unique phenotype: precursor vesicles loosely tethered to the MOP but did not contact its surface. Thus, these mpc54 mutants separate the steps of vesicle association and docking. Using these mpc54 mutants, we determined that recruitment of the Rab GTPase Sec4p, as well as the exocyst components Sec3p and Sec8p, to the precursor vesicles requires vesicle docking to the MOP. This suggests that the MOP promotes membrane formation both by localization of precursor vesicles to a particular site and by recruitment of a second tethering complex, the exocyst, that stimulates downstream events of fusion.
At steady state, most Rho GTPases are bound in the cytosol to Rho Guanine nucleotide Dissociation Inhibitors (RhoGDI) 1. RhoGDIs have generally been considered to passively hold Rho proteins in an inactive state within the cytoplasm. Here we describe an evolutionarily conserved mechanism by which RhoGDI1 controls the homeostasis of Rho proteins in eukaryotic cells. We found that depletion of RhoGDI1 promotes misfolding and degradation of the cytosolic geranylgeranylated pool of Rho GTPases while unexpectedly activating the remaining membrane-bound fraction. Since RhoGDI1 levels are limiting, and Rho proteins compete for binding to RhoGDI1, overexpression of an exogenous Rho GTPase displaces endogenous Rho proteins bound to RhoGDI1, inducing their degradation and inactivation. These results raise important questions about the conclusions drawn from studies that manipulate Rho protein levels. In many cases the response observed may arise not simply from the overexpression per se, but from additional effects on the levels and activity of other Rho GTPases due to competition for binding to RhoGDI1, and may require a re-evaluation of previously published studies that rely exclusively on these techniques.
Synaptotagmin isoforms and mutants altered fusion event frequency and fusion pore transitions. These effects showed a strong correlation with PS binding, but not with SNARE binding. Synaptotagmin-PS interaction thus function in two distinct kinetic steps in Ca2+ triggered exocytosis, and stabilize open fusion pores.
Synaptotagmin (syt) serves as a Ca2+ sensor in the release of neurotransmitters and hormones. This function depends on the ability of syt to interact with other molecules. Syt binds to phosphatidylserine (PS)-containing lipid bilayers as well as to soluble N-ethylmaleimide sensitive factor receptors (SNAREs) and promotes SNARE assembly. All these interactions are regulated by Ca2+, but their specific roles in distinct kinetic steps of exocytosis are not well understood. To explore these questions we used amperometry recording from PC12 cells to investigate the kinetics of exocytosis. Syt isoforms and syt I mutants were overexpressed to perturb syt-PS and syt-SNARE interactions to varying degrees and evaluate the effects on fusion event frequency and the rates of fusion pore transitions. Syt I produced more rapid dilation of fusion pores than syt VII or syt IX, consistent with its role in synchronous synaptic release. Stronger syt-PS interactions were accompanied by a higher frequency of fusion events and more stable fusion pores. By contrast, syt-SNARE interactions and syt-induced SNARE assembly were uncorrelated with rates of exocytosis. This associates the syt-PS interaction with two distinct kinetic steps in Ca2+ triggered exocytosis and supports a role for the syt-PS interaction in stabilizing open fusion pores.
In epithelial cells, Sec3 associates with Exocyst complexes enriched at desmosomes and centrosomes, distinct from Sec6/8 complexes at the apical junctional complex. RNAi-mediated suppression of Sec3 alters trafficking of desmosomal cadherins and impairs desmosome morphology and function, without noticeable effect on adherens junctions.
The Exocyst is a conserved multisubunit complex involved in the docking of post-Golgi transport vesicles to sites of membrane remodeling during cellular processes such as polarization, migration, and division. In mammalian epithelial cells, Exocyst complexes are recruited to nascent sites of cell–cell contact in response to E-cadherin–mediated adhesive interactions, and this event is an important early step in the assembly of intercellular junctions. Sec3 has been hypothesized to function as a spatial landmark for the development of polarity in budding yeast, but its role in epithelial cells has not been investigated. Here, we provide evidence in support of a function for a Sec3-containing Exocyst complex in the assembly or maintenance of desmosomes, adhesive junctions that link intermediate filament networks to sites of strong intercellular adhesion. We show that Sec3 associates with a subset of Exocyst complexes that are enriched at desmosomes. Moreover, we found that membrane recruitment of Sec3 is dependent on cadherin-mediated adhesion but occurs later than that of the known Exocyst components Sec6 and Sec8 that are recruited to adherens junctions. RNA interference-mediated suppression of Sec3 expression led to specific impairment of both the morphology and function of desmosomes, without noticeable effect on adherens junctions. These results suggest that two different exocyst complexes may function in basal–lateral membrane trafficking and will enable us to better understand how exocytosis is spatially organized during development of epithelial plasma membrane domains.
The synaptic vesicle protein synaptotagmin I (Syt I) binds phosphatidylserine (PS) in a Ca2+-dependent manner. This interaction is thought to play a role in exocytosis, but its precise functions remain unclear. To determine potential roles for Syt I-PS binding, we varied the PS content in PC12 cells and liposomes and studied the effects on the kinetics of exocytosis and Syt I binding in parallel. Raising PS produced a steeply nonlinear, saturating increase in Ca2+-triggered fusion, and a graded slowing of the rate of fusion pore dilation. Ca2+-Syt I bound liposomes more tightly as PS content was raised, with a steep increase in binding at low PS, and a further gradual increase at higher PS. These two phases in the PS dependence of Ca2+-dependent Syt I binding to lipid may correspond to the two distinct and opposing kinetic effects of PS on exocytosis. PS influences exocytosis in two ways, enhancing an early step leading to fusion pore opening, and slowing a later step when fusion pores dilate. The possible relevance of these results to Ca2+-triggered Syt I binding is discussed along with other possible roles of PS.
Membrane tethering, the process of mediating the first contact between membranes destined for fusion, requires specialized multisubunit protein complexes and Rab GTPases. In the yeast endolysosomal system, the hexameric HOPS tethering complex cooperates with the Rab7 homolog Ypt7 to promote homotypic fusion at the vacuole, whereas the recently identified homologous CORVET complex acts at the level of late endosomes. Here, we have further functionally characterized the CORVET-specific subunit Vps8 and its relationship to the remaining subunits using an in vivo approach that allows the monitoring of late endosome biogenesis. In particular, our results indicate that Vps8 interacts and cooperates with the activated Rab5 homolog Vps21 to induce the clustering of late endosomal membranes, indicating that Vps8 is the effector subunit of the CORVET complex. This clustering, however, requires Vps3, Vps16, and Vps33 but not the remaining CORVET subunits. These data thus suggest that the CORVET complex is built of subunits with distinct activities and potentially, their sequential assembly could regulate tethering and successive fusion at the late endosomes.
Sec1/Munc18 (SM) proteins bind cognate soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes and stimulate vesicle membrane fusion. Before fusion, vesicles are docked to specific target membranes. Regulation of vesicle docking is attributed to some but not all SM proteins, suggesting specialization of this earlier function. Yeast Sec1p seems to function only after vesicles are docked and SNARE complexes are assembled. Here, we show that yeast Sec1p is required before and after SNARE complex assembly, in support of general requirements for SM proteins in both vesicle docking and fusion. Two classes of sec1 mutants were isolated. Class A mutants are tightly blocked in cell growth and secretion at a step before SNARE complex assembly. Class B mutants have a SNARE complex binding defect, with a range in severity of cell growth and secretion defects. Mapping the mutations onto an SM protein structure implicates a peripheral bundle of helices for the early, docking function and a deep groove, opposite the syntaxin-binding cleft on nSec1/Munc-18, for the interaction between Sec1p and the exocytic SNARE complex.
Glucosidase II (GII) plays a key role in glycoprotein biogenesis in the endoplasmic reticulum (ER). It is responsible for the sequential removal of the two innermost glucose residues from the glycan (Glc3Man9GlcNAc2) transferred to Asn residues in proteins. GII participates in the calnexin/calreticulin cycle; it removes the single glucose unit added to folding intermediates and misfolded glycoproteins by the UDP-Glc:glycoprotein glucosyltransferase. GII is a heterodimer whose α subunit (GIIα) bears the glycosyl hydrolase active site, whereas its β subunit (GIIβ) role is controversial and has been reported to be involved in GIIα ER retention and folding. Here, we report that in the absence of GIIβ, the catalytic subunit GIIα of the fission yeast Schizosaccharomyces pombe (an organism displaying a glycoprotein folding quality control mechanism similar to that occurring in mammalian cells) folds to an active conformation able to hydrolyze p-nitrophenyl α-d-glucopyranoside. However, the heterodimer is required to efficiently deglucosylate the physiological substrates Glc2Man9GlcNAc2 (G2M9) and Glc1Man9GlcNAc2 (G1M9). The interaction of the mannose 6-phosphate receptor homologous domain present in GIIβ and mannoses in the B and/or C arms of the glycans mediates glycan hydrolysis enhancement. We present evidence that also in mammalian cells GIIβ modulates G2M9 and G1M9 trimming.
Obscurin is a large (∼800-kDa), modular protein of striated muscle that concentrates around the M-bands and Z-disks of each sarcomere, where it is well positioned to sense contractile activity. Obscurin contains several signaling domains, including a rho-guanine nucleotide exchange factor (rhoGEF) domain and tandem pleckstrin homology domain, consistent with a role in rho signaling in muscle. We investigated the ability of obscurin's rhoGEF domain to interact with and activate small GTPases. Using a combination of in vitro and in vivo approaches, we found that the rhoGEF domain of obscurin binds selectively to rhoA, and that rhoA colocalizes with obscurin at the M-band in skeletal muscle. Other small GTPases, including rac1 and cdc42, neither associate with the rhoGEF domain of obscurin nor concentrate at the level of the M-bands. Furthermore, overexpression of the rhoGEF domain of obscurin in adult skeletal muscle selectively increases rhoA expression and activity in this tissue. Overexpression of obscurin's rhoGEF domain and its effects on rhoA alter the expression of rho kinase and citron kinase, both of which can be activated by rhoA in other tissues. Injuries to rodent hindlimb muscles caused by large-strain lengthening contractions increases rhoA activity and displaces it from the M-bands to Z-disks, similar to the effects of overexpression of obscurin's rhoGEF domain. Our results suggest that obscurin's rhoGEF domain signals at least in part by inducing rhoA expression and activation, and altering the expression of downstream kinases in vitro and in vivo.
Myo4p, a single-headed and nonprocessive class V myosin in budding yeast, transports >20 different mRNAs asymmetrically to the bud. Here, we determine the features of the Myo4p motor that are necessary for correct localization of ASH1 mRNA to the daughter cell, a process that also requires the adapter protein She3p and the dimeric mRNA-binding protein She2p. The rod region of Myo4p, but not the globular tail, is essential for correct localization of ASH1 mRNA, confirming that the rod contains the primary binding site for She3p. The requirement for both the rod region and She3p can be bypassed by directly coupling the mRNA-binding protein She2p to Myo4p. ASH1 mRNA was also correctly localized when one motor was bound per dimeric She2p, or when two motors were joined together by a leucine zipper. Because multiple mRNAs are cotransported to the bud, it is likely that this process involves multiple motor transport regardless of the number of motors per zip code. Our results show that the most important feature for correct localization is the retention of coupling between all the members of the complex (Myo4p–She3p–She2p–ASH1 mRNA), which is aided by She3p being a tightly bound subunit of Myo4p.
The yeast Smf1p Nramp manganese transporter is posttranslationally regulated by environmental manganese. Smf1p is stabilized at the cell surface with manganese starvation, but is largely degraded in the vacuole with physiological manganese through a mechanism involving the Rsp5p adaptor complex Bsd2p/Tre1p/Tre2p. We now describe an additional level of Smf1p regulation that occurs with toxicity from manganese, but not other essential metals. This regulation is largely Smf1p-specific. As with physiological manganese, toxic manganese triggers vacuolar degradation of Smf1p by trafficking through the multivesicular body. However, regulation by toxic manganese does not involve Bsd2p/Tre1p/Tre2p. Toxic manganese triggers both endocytosis of cell surface Smf1p and vacuolar targeting of intracellular Smf1p through the exocytic pathway. Notably, the kinetics of vacuolar targeting for Smf1p are relatively slow with toxic manganese and require prolonged exposures to the metal. Down-regulation of Smf1p by toxic manganese does not require transport activity of Smf1p, whereas such transport activity is needed for Smf1p regulation by manganese starvation. Furthermore, the responses to manganese starvation and manganese toxicity involve separate cellular compartments. We provide evidence that manganese starvation is sensed within the lumen of the secretory pathway, whereas manganese toxicity is sensed within an extra-Golgi/cytosolic compartment of the cell.
Septins are conserved, GTP-binding proteins that assemble into higher order structures, including filaments and rings with varied cellular functions. Using four-dimensional quantitative fluorescence microscopy of Ashbya gossypii fungal cells, we show that septins can assemble into morphologically distinct classes of rings that vary in dimensions, intensities, and positions within a single cell. Notably, these different classes coexist and persist for extended times, similar in appearance and behavior to septins in mammalian neurons and cultured cells. We demonstrate that new septin proteins can add through time to assembled rings, indicating that septins may continue to polymerize during ring maturation. Different classes of rings do not arise from the presence or absence of specific septin subunits and ring maintenance does not require the actin and microtubule cytoskeletons. Instead, morphological and behavioral differences in the rings require the Elm1p and Gin4p kinases. This work demonstrates that distinct higher order septin structures form within one cell because of the action of specific kinases.
The exocyst is an essential protein complex required for targeting and fusion of secretory vesicles to sites of exocytosis at the plasma membrane. To study the function of the exocyst complex, we performed a structure-based mutational analysis of the Saccharomyces cerevisiae exocyst subunit Sec6p. Two “patches” of highly conserved residues are present on the surface of Sec6p; mutation of either patch does not compromise protein stability. Nevertheless, replacement of SEC6 with the patch mutants results in severe temperature-sensitive growth and secretion defects. At nonpermissive conditions, although trafficking of secretory vesicles to the plasma membrane is unimpaired, none of the exocyst subunits are polarized. This is consistent with data from other exocyst temperature-sensitive mutants, which disrupt the integrity of the complex. Surprisingly, however, these patch mutations result in mislocalized exocyst complexes that remain intact. Our results indicate that assembly and polarization of the exocyst are functionally separable events, and that Sec6p is required to anchor exocyst complexes at sites of secretion.
In regulated vesicle exocytosis, SNARE protein complexes drive membrane fusion to connect the vesicle lumen with the extracellular space. The triggering of fusion pore formation by Ca2+ is mediated by specific isoforms of synaptotagmin (Syt), which employ both SNARE complex and membrane binding. Ca2+ also promotes fusion pore expansion and Syts have been implicated in this process but the mechanisms involved are unclear. We determined the role of Ca2+-dependent Syt-effector interactions in fusion pore expansion by expressing Syt-1 mutants selectively altered in Ca2+-dependent SNARE binding or in Ca2+-dependent membrane insertion in PC12 cells that lack vesicle Syts. The release of different-sized fluorescent peptide-EGFP vesicle cargo or the vesicle capture of different-sized external fluorescent probes was used to assess the extent of fusion pore dilation. We found that PC12 cells expressing partial loss-of-function Syt-1 mutants impaired in Ca2+-dependent SNARE binding exhibited reduced fusion pore opening probabilities and reduced fusion pore expansion. Cells with gain-of-function Syt-1 mutants for Ca2+-dependent membrane insertion exhibited normal fusion pore opening probabilities but the fusion pores dilated extensively. The results indicate that Syt-1 uses both Ca2+-dependent membrane insertion and SNARE binding to drive fusion pore expansion.
Rab GTPases recruit myosin motors to endocytic compartments, which in turn are required for their motility. However, no Ypt/Rab GTPase has been shown to regulate the motility of exocytic compartments. In yeast, the Ypt31/32 functional pair is required for the formation of trans-Golgi vesicles. The myosin V motor Myo2 attaches to these vesicles through its globular-tail domain (GTD) and mediates their polarized delivery to sites of cell growth. Here, we identify Myo2 as an effector of Ypt31/32 and show that the Ypt31/32–Myo2 interaction is required for polarized secretion. Using the yeast-two hybrid system and coprecipitation of recombinant proteins, we show that Ypt31/32 in their guanosine triphosphate (GTP)-bound form interact directly with Myo2-GTD. The physiological relevance of this interaction is shown by colocalization of the proteins, genetic interactions between their genes, and rescue of the lethality caused by a mutation in the Ypt31/32-binding site of Myo2-GTD through fusion with Ypt32. Furthermore, microscopic analyses show a defective Myo2 intracellular localization in ypt31Δ/32ts and in Ypt31/32-interaction–deficient myo2 mutant cells, as well as accumulation of unpolarized secretory vesicles in the latter mutant cells. Together, these results indicate that Ypt31/32 play roles in both the formation of trans-Golgi vesicles and their subsequent Myo2-dependent motility.
Rho G proteins and their regulators are critical for cytoskeleton organization and cell morphology in all eukaryotes. In the opportunistic pathogen Candida albicans, the Rho G proteins Cdc42 and Rac1 are required for the switch from budding to filamentous growth in response to different stimuli. We show that Dck1, a protein with homology to the Ced-5, Dock180, myoblast city family of guanine nucleotide exchange factors, is necessary for filamentous growth in solid media, similar to Rac1. Our results indicate that Dck1 and Rac1 do not function in the same pathway as the transcription factor Czf1, which is also required for embedded filamentous growth. The conserved catalytic region of Dck1 is required for such filamentous growth, and in vitro this region directly binds a Rac1 mutant, which mimics the nucleotide-free state. In vivo overexpression of a constitutively active Rac1 mutant, but not wild-type Rac1, in a dck1 deletion mutant restores filamentous growth. These results indicate that the Dock180 guanine nucleotide exchange factor homologue, Dck1 activates Rac1 during invasive filamentous growth. We conclude that specific exchange factors, together with the G proteins they activate, are required for morphological changes in response to different stimuli.
Mutations in the protein SIMPLE account for the rare autosomal-dominant demyelination in type 1C CMT patients (CMT1C). SIMPLE plays a role in the production of exosomes. Dysregulated endosomal trafficking and changes in exosome-mediated intercellular communications might account for CMT1C molecular pathogenesis.
Charcot–Marie–Tooth (CMT) disease is an inherited neurological disorder. Mutations in the small integral membrane protein of the lysosome/late endosome (SIMPLE) account for the rare autosomal-dominant demyelination in CMT1C patients. Understanding the molecular basis of CMT1C pathogenesis is impeded, in part, by perplexity about the role of SIMPLE, which is expressed in multiple cell types. Here we show that SIMPLE resides within the intraluminal vesicles of multivesicular bodies (MVBs) and inside exosomes, which are nanovesicles secreted extracellularly. Targeting of SIMPLE to exosomes is modulated by positive and negative regulatory motifs. We also find that expression of SIMPLE increases the number of exosomes and secretion of exosome proteins. We engineer a point mutation on the SIMPLE allele and generate a physiological mouse model that expresses CMT1C-mutated SIMPLE at the endogenous level. We find that CMT1C mouse primary embryonic fibroblasts show decreased number of exosomes and reduced secretion of exosome proteins, in part due to improper formation of MVBs. CMT1C patient B cells and CMT1C mouse primary Schwann cells show similar defects. Together the data indicate that SIMPLE regulates the production of exosomes by modulating the formation of MVBs. Dysregulated endosomal trafficking and changes in the landscape of exosome-mediated intercellular communications may place an overwhelming burden on the nervous system and account for CMT1C molecular pathogenesis.
Epithelial differentiation involves the generation of luminal surfaces and of a noncentrosomal microtubule (MT) network aligned along the polarity axis. Columnar epithelia (e.g., kidney, intestine, and Madin-Darby canine kidney [MDCK] cells) generate apical lumina and orient MT vertically, whereas liver epithelial cells (hepatocytes and WIFB9 cells) generate lumina at cell–cell contact sites (bile canaliculi) and orient MTs horizontally. We report that knockdown or inhibition of the mammalian orthologue of Caenorhabditis elegans Par-1 (EMK1 and MARK2) during polarization of cultured MDCK and WIFB9 cells prevented development of their characteristic lumen and nonradial MT networks. Conversely, EMK1 overexpression induced the appearance of intercellular lumina and horizontal MT arrays in MDCK cells, making EMK1 the first known candidate to regulate the developmental branching decision between hepatic and columnar epithelial cells. Our experiments suggest that EMK1 primarily promotes reorganization of the MT network, consistent with the MT-regulating role of this gene product in other systems, which in turn controls lumen formation and position.
EMK1; MARK2; MDCK; WIFB; apical surface
The Rho family GTPase Cdc42 is a key regulator of cell polarity and cytoskeletal organization in eukaryotic cells. In yeast, the role of Cdc42 in polarization of cell growth includes polarization of the actin cytoskeleton, which delivers secretory vesicles to growth sites at the plasma membrane. We now describe a novel temperature-sensitive mutant, cdc42-6, that reveals a role for Cdc42 in docking and fusion of secretory vesicles that is independent of its role in actin polarization. cdc42-6 mutants can polarize actin and deliver secretory vesicles to the bud, but fail to fuse those vesicles with the plasma membrane. This defect is manifested only during the early stages of bud formation when growth is most highly polarized, and appears to reflect a requirement for Cdc42 to maintain maximally active exocytic machinery at sites of high vesicle throughput. Extensive genetic interactions between cdc42-6 and mutations in exocytic components support this hypothesis, and indicate a functional overlap with Rho3, which also regulates both actin organization and exocytosis. Localization data suggest that the defect in cdc42-6 cells is not at the level of the localization of the exocytic apparatus. Rather, we suggest that Cdc42 acts as an allosteric regulator of the vesicle docking and fusion apparatus to provide maximal function at sites of polarized growth.
Cdc42; Rho; GTPases; exocytosis; cell polarity