Myosin- and Rab-interacting protein is not a classic receptor for MyoVa on large, dense-core secretory granules (SGs), but it aids in PKA-dependent phosphorylation of MyoVa-associated proteins on SGs in endocrine and neuroendocrine cells.
Myosin- and Rab-interacting protein (MyRIP), which belongs to the protein kinase A (PKA)–anchoring family, is implicated in hormone secretion. However, its mechanism of action is not fully elucidated. Here we investigate the role of MyRIP in myosin Va (MyoVa)-dependent secretory granule (SG) transport and secretion in pancreatic beta cells. These cells solely express the brain isoform of MyoVa (BR-MyoVa), which is a key motor protein in SG transport. In vitro pull-down, coimmunoprecipitation, and colocalization studies revealed that MyRIP does not interact with BR-MyoVa in glucose-stimulated pancreatic beta cells, suggesting that, contrary to previous notions, MyRIP does not link this motor protein to SGs. Glucose-stimulated insulin secretion is augmented by incretin hormones, which increase cAMP levels and leads to MyRIP phosphorylation, its interaction with BR-MyoVa, and phosphorylation of the BR-MyoVa receptor rabphilin-3A (Rph-3A). Rph-3A phosphorylation on Ser-234 was inhibited by small interfering RNA knockdown of MyRIP, which also reduced cAMP-mediated hormone secretion. Demonstrating the importance of this phosphorylation, nonphosphorylatable and phosphomimic Rph-3A mutants significantly altered hormone release when PKA was activated. These data suggest that MyRIP only forms a functional protein complex with BR-MyoVa on SGs when cAMP is elevated and under this condition facilitates phosphorylation of SG-associated proteins, which in turn can enhance secretion.
OCA2 is used as a model melanosome cargo protein to define primary sequence elements required for acidic dileucine–motif binding to adaptors AP-1 and AP-3. OCA2 must bind to AP-3 for melanosome localization. BLOC-1 is also required and thus can cooperate with either adaptor for cargo delivery to lysosome-related organelles.
Cell types that generate unique lysosome-related organelles (LROs), such as melanosomes in melanocytes, populate nascent LROs with cargoes that are diverted from endosomes. Cargo sorting toward melanosomes correlates with binding via cytoplasmically exposed sorting signals to either heterotetrameric adaptor AP-1 or AP-3. Some cargoes bind both adaptors, but the relative contribution of each adaptor to cargo recognition and their functional interactions with other effectors during transport to melanosomes are not clear. Here we exploit targeted mutagenesis of the acidic dileucine–based sorting signal in the pigment cell–specific protein OCA2 to dissect the relative roles of AP-1 and AP-3 in transport to melanosomes. We show that binding to AP-1 or AP-3 depends on the primary sequence of the signal and not its position within the cytoplasmic domain. Mutants that preferentially bound either AP-1 or AP-3 each trafficked toward melanosomes and functionally complemented OCA2 deficiency, but AP-3 binding was necessary for steady-state melanosome localization. Unlike tyrosinase, which also engages AP-3 for optimal melanosomal delivery, both AP-1– and AP-3–favoring OCA2 variants required BLOC-1 for melanosomal transport. These data provide evidence for distinct roles of AP-1 and AP-3 in OCA2 transport to melanosomes and indicate that BLOC-1 can cooperate with either adaptor during cargo sorting to LROs.
Defining the cellular pathway(s) by which LR11 modulates amyloid-β peptide production is critical to understanding how changes in LR11 expression affect the progression of Alzheimer's disease. These results suggest that endocytic traffic of LR11, facilitated by GGA1, enhances LR11 modulation of amyloid precursor protein processing in a nonamyloidogenic manner.
Proteolytic processing of the amyloid-β precursor protein (APP) and generation of amyloid-β peptide (Aβ) are key events in Alzheimer's disease (AD) pathogenesis. Cell biological and genetic evidence has implicated the low-density lipoprotein and sorting receptor LR11/SorLA in AD through mechanisms related to APP and Aβ production. Defining the cellular pathway(s) by which LR11 modulates Aβ production is critical to understanding how changes in LR11 expression affect the development of Aβ pathology in AD progression. We report that the LR11 ectodomain is required for LR11-mediated reduction of Aβ and that mutagenesis of the LR11 Golgi-localizing, γ-adaptin ear homology domain, ADP-ribosylation factor (GGA)-binding motif affects the endosomal distribution of LR11, as well as LR11's effects on APP traffic and Aβ production. Targeted small interfering RNA (siRNA) knockdown studies of GGA1, GGA2, and GGA3 indicate a surprising degree of specificity toward GGA1, suggesting that GGA1 is a candidate regulator of LR11 traffic. Additional siRNA knockdown experiments reveal that GGA1 is necessary for both LR11 and β-site APP-cleaving enzyme-1 (BACE1) modulation of APP processing to Aβ. Mutagenesis of BACE1 serine 498 to alanine enhances BACE1 targeting to LR11-positive compartments and nullifies LR11-mediated reduction of Aβ. On basis of these results, we propose that GGA1 facilitates LR11 endocytic traffic and that LR11 modulates Aβ levels by promoting APP traffic to the endocytic recycling compartment.
Signaling by phosphatidylinositol 4-kinases (PI4Ks) in the Golgi apparatus controls lipid homeostasis and protein-sorting pathways. Signaling is shown to be terminated on the medial cisterna by a complex of a PI4K effector, Vps74, and Sac1, the major PtdIns4P phosphatase in the cell.
In the Golgi apparatus, lipid homeostasis pathways are coordinated with the biogenesis of cargo transport vesicles by phosphatidylinositol 4-kinases (PI4Ks) that produce phosphatidylinositol 4-phosphate (PtdIns4P), a signaling molecule that is recognized by downstream effector proteins. Quantitative analysis of the intra-Golgi distribution of a PtdIns4P reporter protein confirms that PtdIns4P is enriched on the trans-Golgi cisterna, but surprisingly, Vps74 (the orthologue of human GOLPH3), a PI4K effector required to maintain residence of a subset of Golgi proteins, is distributed with the opposite polarity, being most abundant on cis and medial cisternae. Vps74 binds directly to the catalytic domain of Sac1 (KD = 3.8 μM), the major PtdIns4P phosphatase in the cell, and PtdIns4P is elevated on medial Golgi cisternae in cells lacking Vps74 or Sac1, suggesting that Vps74 is a sensor of PtdIns4P level on medial Golgi cisternae that directs Sac1-mediated dephosphosphorylation of this pool of PtdIns4P. Consistent with the established role of Sac1 in the regulation of sphingolipid biosynthesis, complex sphingolipid homeostasis is perturbed in vps74Δ cells. Mutant cells lacking complex sphingolipid biosynthetic enzymes fail to properly maintain residence of a medial Golgi enzyme, and cells lacking Vps74 depend critically on complex sphingolipid biosynthesis for growth. The results establish additive roles of Vps74-mediated and sphingolipid-dependent sorting of Golgi residents.
Sorting of secretory cargo from the Golgi remains an elusive process. Previously a role was identified for cofilin and the Ca2+ATPase SPCA1 in sorting of secretory cargo from the Golgi of mammalian cells. Now it is shown that the yeast orthologues cofilin and Pmr1 are also required for sorting of selective secretory cargo at the Golgi in yeast.
The mechanism of cargo sorting at the trans-Golgi network (TGN) for secretion is poorly understood. We previously reported the involvement of the actin-severing protein cofilin and the Ca2+ ATPase secretory pathway calcium ATPase 1 (SPCA1) in the sorting of soluble secretory cargo at the TGN in mammalian cells. Now we report that cofilin in yeast is required for export of selective secretory cargo at the late Golgi membranes. In cofilin mutant (cof1-8) cells, the cell wall protein Bgl2 was secreted at a reduced rate and retained in a late Golgi compartment, whereas the plasma membrane H+ ATPase Pma1, which is transported in the same class of carriers, reached the cell surface. In addition, sorting of carboxypeptidase Y (CPY) to the vacuole was delayed, and CPY was secreted from cof1-8 cells. Loss of the yeast orthologue of SPCA1 (Pmr1) exhibited similar sorting defects and displayed synthetic sickness with cof1-8. In addition, overexpression of PMR1 restored Bgl2 secretion in cof1-8 cells. These findings highlight the conserved role of cofilin and SPCA1/Pmr1 in sorting of the soluble secretory proteins at the TGN/late Golgi membranes in eukaryotes.
An adaptor protein complex, AP-2, is involved in the endocytosis of β-secretase (BACE1) via the clathrin-dependent machinery. Endosomal targeting of either the amyloid precursor protein (APP) and/or BACE1 is expendable for the amyloidogenic processing of APP.
The β-site amyloid precursor protein (APP)–cleaving enzyme 1 (BACE1) is a transmembrane aspartyl protease that catalyzes the proteolytic processing of APP and other plasma membrane protein precursors. BACE1 cycles between the trans-Golgi network (TGN), the plasma membrane, and endosomes by virtue of signals contained within its cytosolic C-terminal domain. One of these signals is the DXXLL-motif sequence DISLL, which controls transport between the TGN and endosomes via interaction with GGA proteins. Here we show that the DISLL sequence is embedded within a longer [DE]XXXL[LI]-motif sequence, DDISLL, which mediates internalization from the plasma membrane by interaction with the clathrin-associated, heterotetrameric adaptor protein 2 (AP-2) complex. Mutation of this signal or knockdown of either AP-2 or clathrin decreases endosomal localization and increases plasma membrane localization of BACE1. Remarkably, internalization-defective BACE1 is able to cleave an APP mutant that itself cannot be delivered to endosomes. The drug brefeldin A reversibly prevents BACE1-catalyzed APP cleavage, ruling out that this reaction occurs in the endoplasmic reticulum (ER) or ER–Golgi intermediate compartment. Taken together, these observations support the notion that BACE1 is capable of cleaving APP in late compartments of the secretory pathway.
Using computational modeling and laser microsurgery, we establish that neither the centrosomal microtubule array nor the Golgi-derived array is solely sufficient for correct Golgi assembly. Only the concerted effort of both MT arrays results in the integral, polarized Golgi complex necessary for polarized trafficking and cell motility.
Assembly of an integral Golgi complex is driven by microtubule (MT)-dependent transport. Conversely, the Golgi itself functions as an unconventional MT-organizing center (MTOC). This raises the question of whether Golgi assembly requires centrosomal MTs or can be self-organized, relying on its own MTOC activity. The computational model presented here predicts that each MT population is capable of gathering Golgi stacks but not of establishing Golgi complex integrity or polarity. In contrast, the concerted effort of two MT populations would assemble an integral, polarized Golgi complex. Indeed, while laser ablation of the centrosome did not alter already-formed Golgi complexes, acentrosomal cells fail to reassemble an integral complex upon nocodazole washout. Moreover, polarity of post-Golgi trafficking was compromised under these conditions, leading to strong deficiency in polarized cell migration. Our data indicate that centrosomal MTs complement Golgi self-organization for proper Golgi assembly and motile-cell polarization.
c-Fos increases the overall synthesis of polyphosphoinositides by an AP-1–independent mechanism involving activation of CDP-diacylglycerol synthase and phosphatidylinositol (PtdIns) 4-kinase II α but not of PtdIns synthase or PtdIns 4-kinase II β. Coimmunoprecipitation and FRET experiments show that c-Fos physically associates only with the enzymes it activates.
The oncoprotein c-Fos is a well-recognized AP-1 transcription factor. In addition, this protein associates with the endoplasmic reticulum and activates the synthesis of phospholipids. However, the mechanism by which c-Fos stimulates the synthesis of phospholipids in general and the specific lipid pathways activated are unknown. Here we show that induction of quiescent cells to reenter growth promotes an increase in the labeling of polyphosphoinositides that depends on the expression of c-Fos. We also investigated whether stimulation by c-Fos of the synthesis of phosphatidylinositol and its phosphorylated derivatives depends on the activation of enzymes of the phosphatidylinositolphosphate biosynthetic pathway. We found that c-Fos activates CDP-diacylglycerol synthase and phosphatidylinositol (PtdIns) 4-kinase II α in vitro, whereas no activation of phosphatidylinositol synthase or of PtdIns 4-kinase II β was observed. Both coimmunoprecipitation and fluorescence resonance energy transfer experiments consistently showed a physical interaction between the N-terminal domain of c-Fos and the enzymes it activates.
Functional characterization of the Golgi-associated retrograde protein (GARP) complex in Caenorhabditis elegans has led to the identification of the conserved metazoan Vps51 subunit. It is found that GARP mutants lead to abnormal lysosomal morphology, GARP subunits interact with a distinct set of Golgi SNAREs, and GARP and GOG complexes show functional overlap.
In yeast the Golgi-associated retrograde protein (GARP) complex is required for tethering of endosome-derived transport vesicles to the late Golgi. It consists of four subunits—Vps51p, Vps52p, Vps53p, and Vps54p—and shares similarities with other multimeric tethering complexes, such as the conserved oligomeric Golgi (COG) and the exocyst complex. Here we report the functional characterization of the GARP complex in the nematode Caenorhabditis elegans. Furthermore, we identified the C. elegans Vps51 subunit, which is conserved in all eukaryotes. GARP mutants are viable but show lysosomal defects. We show that GARP subunits bind specific sets of Golgi SNAREs within the yeast two-hybrid system. This suggests that the C. elegans GARP complex also facilitates tethering as well as SNARE complex assembly at the Golgi. The GARP and COG tethering complexes may have overlapping functions for retrograde endosome-to-Golgi retrieval, since loss of both complexes leads to a synthetic lethal phenotype.
The mechanism of collagen secretion is not completely understood. It is found that cTAGE5 binds to TANGO1, and it is suggested that collagen VII export from the ER is driven by a cTAGE5/TANGO1 complex.
Cutaneous T-cell lymphoma-–associated antigen 5 (cTAGE5), an originally identified tumor antigen, is overexpressed in various cancer cell lines. The cDNA encodes an integral membrane protein containing two coiled-coil motifs and a proline-rich domain. We show that cTAGE5 specifically localizes to the endoplasmic reticulum (ER) exit sites. In addition, cTAGE5 forms a complex with TANGO1 (MIA3), a previously characterized cargo receptor for collagen VII, by the interaction of their coiled-coil motifs. Of interest, cTAGE5, as well as TANGO1, is capable of interacting with the inner-layer coatomer of COPII Sec23/24 complex through their C-terminal proline-rich domains and required for collagen VII secretion. We propose that cTAGE5 acts as a coreceptor of TANGO1 for collagen VII export from the ER.
In search of morphological determinants for the endoplasmic reticulum-Golgi intermediate compartment (ERGIC), we found that a concerted action of Arf1, Arf4, and PLA2G6-A controls the architecture of the ERGIC by regulating tubular carriers. This is predicted to impact the rate of transport and destination of cargos in the ERGIC.
Organelle morphology of the endomembrane system is critical for optimal organelle function. ADP ribosylation factors (Arfs), a family of small GTPases, are required for maintaining the structure of the Golgi and endosomes. What determines the discontinuous nature of the endoplasmic reticulum (ER)–Golgi intermediate compartment (ERGIC) as tubulovesicular clusters is unknown. In search of morphological determinants for the ERGIC, we found that a double knockdown of Arf1+Arf4 induced dynamic ERGIC tubules that connect ERGIC clusters, indicating that the tubules mediated lateral intraERGIC traffic. Tubule formation was inhibited by an antagonist of group VI calcium-independent phospholipase A2 (PLA2G6) and by silencing the A isoform of PLA2G6 (PLA2G6-A). Arf1+Arf4 depletion altered the expression of PLA2G6-A splice variants and relocalized PLA2G6-A from the cytosol to ERGIC clusters and tubules, suggesting that the enzyme became locally active. We show that changes in Arf1 can modulate the activity of PLA2G6-A. We propose that a concerted action of Arf1, Arf4, and PLA2G6-A controls the architecture of the ERGIC in a way that is predicted to impact the rate and possibly the destination of cargos. Our findings have identified key components in the molecular mechanism underlying the regulation of tubules in the ERGIC and uncover tubular carriers as tightly controlled machinery.
The protein Ang2 is shown to be a conserved component of the Golgi-associated Retrograde Protein (GARP) complex in higher eukaryotes. Ang2 participates in retrograde transport from endosomes to the TGN, contributing to the maintenance of acid hydrolase sorting, lysosome function and autophagy.
The Golgi-associated retrograde protein (GARP) complex mediates tethering and fusion of endosome-derived transport carriers to the trans-Golgi network (TGN). In the yeast Saccharomyces cerevisiae, GARP comprises four subunits named Vps51p, Vps52p, Vps53p, and Vps54p. Orthologues of the GARP subunits, except for Vps51p, have been identified in all other eukaryotes. A yeast two-hybrid screen of a human cDNA library yielded a phylogenetically conserved protein, Ang2/Fat-free, which interacts with human Vps52, Vps53 and Vps54. Human Ang2 is larger than yeast Vps51p, but exhibits significant homology in an N-terminal coiled-coil region that mediates assembly with other GARP subunits. Biochemical analyses show that human Ang2, Vps52, Vps53 and Vps54 form an obligatory 1:1:1:1 complex that strongly interacts with the regulatory Habc domain of the TGN SNARE, Syntaxin 6. Depletion of Ang2 or the GARP subunits similarly impairs protein retrieval to the TGN, lysosomal enzyme sorting, endosomal cholesterol traffic¤ and autophagy. These findings indicate that Ang2 is the missing component of the GARP complex in most eukaryotes.
We propose a new active mask algorithm for the segmentation of fluorescence microscope images of punctate patterns. It combines the (a) flexibility offered by active-contour methods, (b) speed offered by multiresolution methods, (c) smoothing offered by multiscale methods, and (d) statistical modeling offered by region-growing methods into a fast and accurate segmentation tool. The framework moves from the idea of the “contour” to that of “inside and outside”, or, masks, allowing for easy multidimensional segmentation. It adapts to the topology of the image through the use of multiple masks. The algorithm is almost invariant under initialization, allowing for random initialization, and uses a few easily tunable parameters. Experiments show that the active mask algorithm matches the ground truth well, and outperforms the algorithm widely used in fluorescence microscopy, seeded watershed, both qualitatively as well as quantitatively.
STX6 is a new component of the CAL complex that regulates the abundance and function of CFTR at the post-ER level. Our results suggest a therapeutic role of STX6 in enhancing rescued ΔF508-CFTR.
The PDZ domain–containing protein CAL mediates lysosomal trafficking and degradation of CFTR. Here we demonstrate the involvement of a CAL-binding SNARE protein syntaxin 6 (STX6) in this process. Overexpression of STX6, which colocalizes and coimmunoprecipitates with CAL, dramatically reduces the steady-state level and stability of CFTR. Conversely, overexpression of a STX6 dominant-negative mutant increases CFTR. Silencing endogenous STX6 increases CFTR but has no effect on ΔTRL-CFTR, which cannot bind to CAL. Silencing CAL eliminates the effect of STX6 on CFTR. Both results suggest a dependence of CAL on STX6 function. Consistent with its Golgi localization, STX6 does not bind to ER-localized ΔF508-CFTR. Silencing STX6 has no effect on ΔF508-CFTR expression. However, overexpression of STX6 coimmunoprecipitates with and reduces temperature-rescued ΔF508-CFTR that escapes ER degradation. Conversely, silencing STX6 enhances the effect of low temperature in rescuing ΔF508-CFTR. Finally, in human bronchial epithelial cells, silencing endogenous STX6 leads to increases in protein levels and Cl− currents of both wild-type and temperature-rescued CFTR. We have identified STX6 as a new component of the CAL complex that regulates the abundance and function of CFTR at the post-ER level. Our results suggest a therapeutic role of STX6 in enhancing rescued ΔF508-CFTR.
Manganism is a disease with no cure. This study identifies a mammalian protein with manganese-sensitive trafficking. The findings provide an important, novel example of regulated sorting under physiological conditions particularly in that a lumenal, rather than cytoplasmic, sequence confers the regulation.
Manganese is an essential element that is also neurotoxic at elevated exposure. However, mechanisms regulating Mn homeostasis in mammalian cells are largely unknown. Because increases in cytosolic Mn induce rapid changes in the localization of proteins involved in regulating intracellular Mn concentrations in yeast, we were intrigued to discover that low concentrations of extracellular Mn induced rapid redistribution of the mammalian cis-Golgi glycoprotein Golgi phosphoprotein of 130 kDa (GPP130) to multivesicular bodies. GPP130 was subsequently degraded in lysosomes. The Mn-induced trafficking of GPP130 occurred from the Golgi via a Rab-7–dependent pathway and did not require its transit through the plasma membrane or early endosomes. Although the cytoplasmic domain of GPP130 was dispensable for its ability to respond to Mn, its lumenal stem domain was required and it had to be targeted to the cis-Golgi for the Mn response to occur. Remarkably, the stem domain was sufficient to confer Mn sensitivity to another cis-Golgi protein. Our results identify the stem domain of GPP130 as a novel Mn sensor in the Golgi lumen of mammalian cells.
This study establishes a role for luminal Ca2+ in ER/Golgi transport organelles and elucidates an effector mechanism involving the EF-hand protein ALG-2 and regulation of COPII coat retention.
The significance and extent of Ca2+ regulation of the biosynthetic secretory pathway have been difficult to establish, and our knowledge of regulatory relationships integrating Ca2+ with vesicle coats and function is rudimentary. Here, we investigated potential roles and mechanisms of luminal Ca2+ in the early secretory pathway. Specific depletion of luminal Ca2+ in living normal rat kidney cells using cyclopiazonic acid (CPA) resulted in the extreme expansion of vesicular tubular cluster (VTC) elements. Consistent with this, a suppressive role for vesicle-associated Ca2+ in COPII vesicle homotypic fusion was demonstrated in vitro using Ca2+ chelators. The EF-hand–containing protein apoptosis-linked gene 2 (ALG-2), previously implicated in the stabilization of sec31 at endoplasmic reticulum exit sites, inhibited COPII vesicle fusion in a Ca2+-requiring manner, suggesting that ALG-2 may be a sensor for the effects of vesicular Ca2+ on homotypic fusion. Immunoisolation established that Ca2+ chelation inhibits and ALG-2 specifically favors residual retention of the COPII outer shell protein sec31 on pre-Golgi fusion intermediates. We conclude that vesicle-associated Ca2+, acting through ALG-2, favors the retention of residual coat molecules that seem to suppress membrane fusion. We propose that in cells, these Ca2+-dependent mechanisms temporally regulate COPII vesicle interactions, VTC biogenesis, cargo sorting, and VTC maturation.
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.
Rabex-5 targets to early endosomes and functions as a guanine nucleotide exchange factor for Rab5. Membrane targeting is critical for Rabex-5 to activate Rab5 on early endosomes in the cell. Here, we report the identification of Rab22 as a binding site on early endosomes for direct recruitment of Rabex-5 and activation of Rab5, establishing a Rab22-Rab5 signaling relay to promote early endosome fusion. Rab22 in guanosine 5′-O-(3-thio)triphosphate-loaded form, but not guanosine diphosphate-loaded form, binds to the early endosomal targeting domain (residues 81-230) of Rabex-5 in pull-down assays. Rabex-5 targets to Rab22-containing early endosomes, and Rab22 knockdown by short hairpin RNA abrogates the membrane targeting of Rabex-5 in the cell. In addition, coexpression of Rab22 and Rab5 shows synergistic enlargement of early endosomes, and this synergy is dependent on Rabex-5, providing further support for the collaboration of the two Rab GTPases in regulation of endosome dynamics. This novel Rab22–Rabex-5–Rab5 cascade is functionally important for the endocytosis and degradation of epidermal growth factor.
Formation of the ribbon-like membrane network of the Golgi apparatus depends on GM130 and GRASP65, but the mechanism is unknown. We developed an in vivo organelle tethering assaying in which GRASP65 was targeted to the mitochondrial outer membrane either directly or via binding to GM130. Mitochondria bearing GRASP65 became tethered to one another, and this depended on a GRASP65 PDZ domain that was also required for GRASP65 self-interaction. Point mutation within the predicted binding groove of the GRASP65 PDZ domain blocked both tethering and, in a gene replacement assay, Golgi ribbon formation. Tethering also required proximate membrane anchoring of the PDZ domain, suggesting a mechanism that orientates the PDZ binding groove to favor interactions in trans. Thus, a homotypic PDZ interaction mediates organelle tethering in living cells.
Shiga-toxin–producing Escherichia coli remain a food-borne health threat. Shiga toxin is endocytosed by intestinal epithelial cells and transported retrogradely through the secretory pathway. It is ultimately translocated to the cytosol where it inhibits protein translation. We found that Shiga toxin transport through the secretory pathway was dependent on the cytoskeleton. Recent studies reveal that Shiga toxin activates signaling pathways that affect microtubule reassembly and dynein-dependent motility. We propose that Shiga toxin alters cytoskeletal dynamics in a way that facilitates its transport through the secretory pathway. We have now found that Rho GTPases regulate the endocytosis and retrograde motility of Shiga toxin. The expression of RhoA mutants inhibited endocytosis of Shiga toxin. Constitutively active Cdc42 or knockdown of the Cdc42-specific GAP, ARHGAP21, inhibited the transport of Shiga toxin to the juxtanuclear Golgi apparatus. The ability of Shiga toxin to stimulate microtubule-based transferrin transport also required Cdc42 and ARHGAP21 function. Shiga toxin addition greatly decreases the levels of active Cdc42-GTP in an ARHGAP21-dependent manner. We conclude that ARHGAP21 and Cdc42-based signaling regulates the dynein-dependent retrograde transport of Shiga toxin to the Golgi apparatus.
The endoplasmic reticulum (ER) of animal cells is a single, dynamic, and continuous membrane network of interconnected cisternae and tubules spread out throughout the cytosol in direct contact with the nuclear envelope. During mitosis, the nuclear envelope undergoes a major rearrangement, as it rapidly partitions its membrane-bound contents into the ER. It is therefore of great interest to determine whether any major transformation in the architecture of the ER also occurs during cell division. We present structural evidence, from rapid, live-cell, three-dimensional imaging with confirmation from high-resolution electron microscopy tomography of samples preserved by high-pressure freezing and freeze substitution, unambiguously showing that from prometaphase to telophase of mammalian cells, most of the ER is organized as extended cisternae, with a very small fraction remaining organized as tubules. In contrast, during interphase, the ER displays the familiar reticular network of convolved cisternae linked to tubules.
Adrenal medullary chromaffin cells are innervated by the sympathetic splanchnic nerve and translate graded sympathetic firing into a differential hormonal exocytosis. Basal sympathetic firing elicits a transient kiss-and-run mode of exocytosis and modest catecholamine release, whereas elevated firing under the sympathetic stress response results in full granule collapse to release catecholamine and peptide transmitters into the circulation. Previous studies have shown that rearrangement of the cell actin cortex regulates the mode of exocytosis. An intact cortex favors kiss-and-run exocytosis, whereas disrupting the cortex favors the full granule collapse mode. Here, we investigate the specific roles of two actin-associated proteins, myosin II and myristoylated alanine-rich C-kinase substrate (MARCKS) in this process. Our data demonstrate that MARCKS phosphorylation under elevated cell firing is required for cortical actin disruption but is not sufficient to elicit peptide transmitter exocytosis. Our data also demonstrate that myosin II is phospho-activated under high stimulation conditions. Inhibiting myosin II activity prevented disruption of the actin cortex, full granule collapse, and peptide transmitter release. These results suggest that phosphorylation of both MARCKS and myosin II lead to disruption of the actin cortex. However, myosin II, but not MARCKS, is required for the activity-dependent exocytosis of the peptide transmitters.
Mutations in the FGD1 gene are responsible for the X-linked disorder known as faciogenital dysplasia (FGDY). FGD1 encodes a guanine nucleotide exchange factor that specifically activates the GTPase Cdc42. In turn, Cdc42 is an important regulator of membrane trafficking, although little is known about FGD1 involvement in this process. During development, FGD1 is highly expressed during bone growth and mineralization, and therefore a lack of the functional protein leads to a severe phenotype. Whether the secretion of proteins, which is a process essential for bone formation, is altered by mutations in FGD1 is of great interest. We initially show here that FGD1 is preferentially associated with the trans-Golgi network (TGN), suggesting its involvement in export of proteins from the Golgi. Indeed, expression of a dominant-negative FGD1 mutant and RNA interference of FGD1 both resulted in a reduction in post-Golgi transport of various cargoes (including bone-specific proteins in osteoblasts). Live-cell imaging reveals that formation of post-Golgi transport intermediates directed to the cell surface is inhibited in FGD1-deficient cells, apparently due to an impairment of TGN membrane extension along microtubules. These effects depend on FGD1 regulation of Cdc42 activation and its association with the Golgi membranes, and they may contribute to FGDY pathogenesis.
Peri-centrosomal positioning of the mammalian Golgi apparatus is known to involve microtubule-based motility, but its importance for cellular physiology is a major unanswered question. Here, we identify golgin-160 and GMAP210 as proteins required for centripetal motility of Golgi membranes. In the absence of either golgin, peri-centrosomal positioning of the Golgi apparatus was disrupted while the cytoskeleton remained intact. Although secretion persisted with normal kinetics, it was evenly distributed in response to wounding rather than directed to the wound edge. Strikingly, these cells also completely failed to polarize. Further, directionally persistent cell migration was inhibited such that wound closure was impaired. These findings not only reveal novel roles for golgin-160 and GMAP210 in conferring membrane motility but also indicate that Golgi positioning has an active role in directed secretion, cell polarity, and wound healing.
Oculocutaneous albinism type 2 is caused by defects in the gene OCA2, encoding a pigment cell-specific, 12-transmembrane domain protein with homology to ion permeases. The function of the OCA2 protein remains unknown, and its subcellular localization is under debate. Here, we show that endogenous OCA2 in melanocytic cells rapidly exits the endoplasmic reticulum (ER) and thus does not behave as a resident ER protein. Consistently, exogenously expressed OCA2 localizes within melanocytes to melanosomes, and, like other melanosomal proteins, localizes to lysosomes when expressed in nonpigment cells. Mutagenized OCA2 transgenes stimulate melanin synthesis in OCA2-deficient cells when localized to melanosomes but not when specifically retained in the ER, contradicting a proposed primary function for OCA2 in the ER. Steady-state melanosomal localization requires a conserved consensus acidic dileucine-based sorting motif within the cytoplasmic N-terminal region of OCA2. A second dileucine signal within this region confers steady-state lysosomal localization in melanocytes, suggesting that OCA2 might traverse multiple sequential or parallel trafficking routes. The two dileucine signals physically interact in a differential manner with cytoplasmic adaptors known to function in trafficking other proteins to melanosomes. We conclude that OCA2 is targeted to and functions within melanosomes but that residence within melanosomes may be regulated by secondary or alternative targeting to lysosomes.