Protein kinase D regulates the fission of cell surface destined transport carriers from the trans-Golgi network

When a kinase inactive form of Protein Kinase D (PKD-K618N) was expressed in HeLa cells, it localized to the trans-Golgi network (TGN) and caused extensive tubulation. Cargo that was destined for the plasma membrane was found in PKD-K618N-containing tubes but the tubes did not detach from the TGN. As a result, the transfer of cargo from TGN to the plasma membrane was inhibited. We have also demonstrated the formation and subsequent detachment of cargo-containing tubes from the TGN in cells stably expressing low levels of PKD-K618N. Our results suggest that PKD regulates the fission from the TGN of transport carriers that are en route to the cell surface.     

PKD Regulates Membrane Fission to Generate TGN to Cell Surface Transport Carriers

The serine/threonine protein kinase D (PKD) is recruited to the trans-Golgi network (TGN) by binding diacylglycerol (DAG) and the ARF1 GTPase. PKD, at the TGN, promotes the production of phosphatidylinositol-4 phosphate (PI4P) by activating the lipid kinase phophatidylinositol 4-kinase IIIß (PI4KIIIß). PI4P recruits proteins such as oxysterol-binding protein 1 (OSBP) and ceramide transport protein (CERT) that control sphingolipid and sterol levels at the TGN. CERT mediated transport of ceramide to the TGN, we suggest, is used for increasing the local production and concentration of DAG. Once the crucial concentration of DAG is achieved, OSBP and CERT dissociate from the TGN on phosphorylation by PKD and DAG is sequentially converted into phosphatidic acid (PA) and lyso-PA (LPA). Therefore, the net effect of the activated PKD at the TGN is the sequential production of the modified lipids DAG, PA, and LPA that are necessary for membrane fission to generate cell surface specific transport carriers.     

Rab13 regulates membrane trafficking between TGN and recycling endosomes in polarized epithelial cells

To maintain polarity, epithelial cells continuously sort transmembrane proteins to the apical or basolateral membrane domains during biosynthetic delivery or after internalization. During biosynthetic delivery, some cargo proteins move from the trans-Golgi network (TGN) into recycling endosomes (RE) before being delivered to the plasma membrane. However, proteins that regulate this transport step remained elusive. In this study, we show that Rab13 partially colocalizes with TGN38 at the TGN and transferrin receptors in RE. Knockdown of Rab13 with short hairpin RNA in human bronchial epithelial cells or overexpression of dominant-active or dominant-negative alleles of Rab13 in Madin-Darby canine kidney cells disrupts TGN38/46 localization at the TGN. Moreover, overexpression of Rab13 mutant alleles inhibits surface arrival of proteins that move through RE during biosynthetic delivery (vesicular stomatitis virus glycoprotein [VSVG], A-VSVG, and LDLR-CT27). Importantly, proteins using a direct route from the TGN to the plasma membrane are not affected. Thus, Rab13 appears to regulate membrane trafficking between TGN and RE.     

Shaping tubular carriers for intracellular membrane transport

The particular compositions of the intracellular membrane organelles rely on the proteins and lipids received frequently through membrane trafficking. The delivery of these molecules is driven by the membrane-bound organelles known as transport carriers (TCs). Advanced microscopy approaches have revealed that TC morphology ranges from small vesicles to complex tubular membrane structures. These tubular TCs (TTCs) support effectively both sorting and transport events within the biosynthetic and endocytic pathways, while a coherent picture of the processes that define the formation and further fate of TTCs is still missing. Here, we present an overview of the mechanisms operating during the TTC life cycle, as well as of the emerging role of tubular carriers in different intracellular transport routes.     

Localization of SMAP2 to the TGN and its function in the regulation of TGN protein transport

SMAP2 is an Arf GTPase-activating protein that is located and functions on early endosome membranes. In the present study, the trans-Golgi network (TGN) was verified as an additional site of SMAP2 localization based on its co-localization with various TGN-marker proteins. Mutation of specific stretches of basic amino acid residues abolished the TGN-localization of SMAP2. Over-expression of wild-type SMAP2, but not of the mutated SMAP2, inhibited the transport of vesicular stomatitis virus-G protein from the TGN to the plasma membrane. In contrast, this transport was enhanced in SMAP2 (-/-) cells characterized by increased levels of the activated form of Arf. SMAP2 therefore belongs to an ArfGAP subtype that resides on the TGN and functions as a negative regulator of vesicle budding from the organelle.     

Rab6 regulates transport and targeting of exocytotic carriers

Constitutive exocytosis delivers newly synthesized proteins, lipids, and other molecules from the Golgi apparatus to the cell surface. This process is mediated by vesicles, which bud off the trans-Golgi network, move along cytoskeletal filaments, and fuse with the plasma membrane. Here, we show that the small GTPase Rab6 marks exocytotic vesicles and, together with the microtubule plus-end-directed motor kinesin-1, stimulates their processive microtubule-based transport to the cell periphery. Furthermore, Rab6 directs targeting of secretory vesicles to plasma-membrane sites enriched in the cortical protein ELKS, a known Rab6 binding partner. Our data demonstrate that although Rab6 is not essential for secretion, it controls the organization of exocytosis within the cellular space.     

Cdc42 regulates multiple membrane traffic events in fission yeast

Fission yeast Cdc42 regulates polarized growth and is involved in For3 formin activation and actin cable assembly. We show here that a thermosensitive strain carrying the cdc42L160S allele has membrane traffic defects independent of the actin cable defects. This strain has decreased acid phosphatase (AP) secretion, intracellular accumulation of vesicles and fragmentation of vacuoles. In addition, the exocyst is not localized to the tips of these cells. Overproduction of the scaffold protein Pob1 suppressed cdc42L160S thermosensitive growth and restored exocyst localization and AP secretion. The GTPase Rho3 also suppressed cdc42L160S thermosensitivity, restored exocyst localization and AP secretion. However, Rho3 did not restore the actin cables in these cells as Pob1 does. Similarly, overexpression of psy1(+) , coding a syntaxin (t-SNARE) homolog, or of ypt2(+) , coding an SEC4 homolog in fission yeast, rescued growth at high temperature but did not restore actin cables, nor the exocyst-polarized localization. cdc42L160S cells also have defects in vacuole formation that were rescued by Pob1, Rho3 and Psy1. All together, we propose that Cdc42 and the scaffold Pob1 are required for membrane trafficking and fusion, contributing to polarized secretion, endosome recycling, vacuole formation and growth.     

PKD is recruited to sites of actin remodelling at the leading edge and negatively regulates cell migration

Protein kinase D (PKD) has been implicated in the regulation of cell shape, adhesion, and migration. At the leading edge of migrating cells active PKD co-localizes with F-actin, Arp3 and cortactin. Platelet derived growth factor (PDGF) activates PKD and recruits the kinase to the leading edge, suggesting a role for PKD in actin remodelling. In support of this, PKD directly interacts with F-actin and phosphorylates cortactin in vitro. Interference with PKD function by overexpression of a dominant negative PKD or by PKD-specific siRNA enhanced cell migration, whereas cells overexpressing PKD wild type displayed reduced migratory potential. Taken together, these data reveal a negative regulatory function of PKD in cell migration.     

N-cadherin-catenin complexes form prior to cleavage of the proregion and transport to the plasma membrane

Cadherins are calcium-dependent glycoproteins that function as cell-cell adhesion molecules and are linked to the actin cytoskeleton via catenins. Newly synthesized cadherins contain a prosequence that must be proteolytically removed to generate a functional adhesion molecule. The goal of this study was to examine the proteolytic processing of N-cadherin and the assembly of the cadherin-catenin complex in cells that express endogenous N-cadherin. A monoclonal antibody specific for the proregion of human N-cadherin was generated and used to examine N-cadherin processing. Our data show that newly synthesized proN-cadherin is phosphorylated and proteolytically processed prior to transport to the plasma membrane. In addition, we show that beta-catenin and plakoglobin associate only with phosphorylated proN-cadherin, whereas p120(ctn) can associate with both phosphorylated and non-phosphorylated proN-cadherin. Immunoprecipitations using anti-proN-cadherin showed that cadherin-catenin complexes are assembled prior to localization at the plasma membrane. These data suggest that a core N-cadherin-catenin complex assembles in the endoplasmic reticulum or Golgi compartment and is transported to the plasma membrane where linkage to the actin cytoskeleton can be established.     

ER-to-Golgi transport: form and formation of vesicular and tubular carriers

The transport of proteins and lipids between the endoplasmic reticulum and Golgi apparatus is initiated by the collection of secretory cargo from within the lumen of the endoplasmic reticulum. Subsequently, transport carriers are formed that bud from this membrane and are transported to, and subsequently merge with, the Golgi. The principle driving force behind the budding process is the multi-subunit coat protein complex, COPII. A considerable amount of information is now available regarding the molecular mechanisms by which COPII components operate together to drive cargo selection and transport carrier formation. In contrast, the precise nature of the transport carriers formed is still a matter of considerable debate. Vesicular and tubular carriers have been characterized that are, or in other cases are not, coated with the COPII complex. Here, we seek to integrate much of the data surrounding this topic and try to understand the mechanisms by which vesicular and/or tubular carriers might be generated.     

ADF/cofilin regulates secretory cargo sorting at the TGN via the Ca2+ ATPase SPCA1

1. Download : Download high-res image (239KB)
2. Download : Download full-size image

Highlights? ADF/cofilin localizes to the TGN and binds SPCA1 in an actin-dependent manner ? Dynamic actin regulates the activity of the Ca²⁺ pump SPCA1 ? ADF/cofilin facilitates secretory cargo sorting in a Ca²⁺-dependent manner     

CtBP3/BARS drives membrane fission in dynamin-independent transport pathways

Membrane fission is a fundamental step in membrane transport. So far, the only fission protein machinery that has been implicated in in vivo transport involves dynamin, and functions in several, but not all, transport pathways. Thus, other fission machineries may exist. Here, we report that carboxy-terminal binding protein 3/brefeldin A-ribosylated substrate (CtBP3/BARS) controls fission in basolateral transport from the Golgi to the plasma membrane and in fluid-phase endocytosis, whereas dynamin is not involved in these steps. Conversely, CtBP3/BARS protein is inactive in apical transport to the plasma membrane and in receptor-mediated endocytosis, both steps being controlled by dynamin. This indicates that CtBP3/BARS controls membrane fission in endocytic and exocytic transport pathways, distinct from those that require dynamin.     

rab4 regulates transport to the apical plasma membrane in Madin-Darby canine kidney cells.

The small GTPase rab4 is associated with early endosomes and regulates membrane recycling in fibroblasts. rab4 is present in epithelial cells; however, neither its localization nor function has been established in this cell type. We transfected Madin-Darby canine kidney cells with rab4, the GTPase-deficient mutant rab4Q67L, and the dominant negative mutant rab4S22N that poorly binds guanine nucleotides. Confocal immunofluorescence microscopy showed that rab4 was concentrated on internal structures at the lateral side of the cell around the nucleus. Quantitative immunoelectron microscopy revealed that the majority of rab4 was localized in the upper third of the cytoplasm. In cell surface binding experiments with (125)I-transferrin, we found a redistribution of transferrin receptor from the basolateral to the apical plasma membrane in cells expressing rab4 and rab4Q67L. After accumulation of transferrin at 16 degrees C in basolateral early endosomes, rab4 and rab4Q67L increased the amount of apically targeted transferrin receptor. A qualitatively similar effect was obtained in control cells treated with brefeldin A. The effects of brefeldin A and rab4 on apical targeting of transferrin receptor were not additive, suggesting that brefeldin A and rab4 may act in the same transport pathway from common endosomes.     

Niemann-Pick C1 protein regulates cholesterol transport to the trans-Golgi network and plasma membrane caveolae

The Niemann-Pick C1 (NPC1) protein regulates cholesterol transport from late endosomes-lysosomes to other intracellular compartments. In this article, cholesterol transport to caveolin-1 and caveolin-2 containing compartments, such as the trans-Golgi network (TGN) and plasma membrane caveolae, was examined in normal (NPC+/+), NPC heterozygous (NPC+/-), and NPC homozygous (NPC-/-) human fibroblasts. The expression and distribution of NPC1 in each cell type were similar, and characterized by a finely dispersed, granular staining pattern. The expression of caveolin-1 and caveolin-2 was increased in NPC+/- and NPC-/- fibroblasts, although the distribution in each cell type was similar and characterized by predominant staining of the TGN and plasma membrane. The TGN in NPC+/+ fibroblasts was relatively cholesterol-enriched, whereas the TGN in NPC+/- and NPC-/- fibroblasts was partially or completely cholesterol-deficient, respectively. Consistent with studies demonstrating the transport of cholesterol from the TGN to plasma membrane caveolae, the concentration of cholesterol in plasma membrane caveolae isolated from NPC+/- and NPC-/- fibroblasts was significantly decreased, even though the total concentration of plasma membrane cholesterol in each cell type was similar.These studies demonstrate that NPC1 regulates cholesterol transport to caveolin-1 and caveolin-2 containing compartments such as the TGN and plasma membrane caveolae.     

PI4P/PS countertransport by ORP10 at ER–endosome membrane contact sites regulates endosome fission

Membrane contact sites (MCSs) serve as a zone for nonvesicular lipid transport by oxysterol-binding protein (OSBP)-related proteins (ORPs). ORPs mediate lipid countertransport, in which two distinct lipids are transported counterdirectionally. How such lipid countertransport controls specific biological functions, however, remains elusive. We report that lipid countertransport by ORP10 at ER–endosome MCSs regulates retrograde membrane trafficking. ORP10, together with ORP9 and VAP, formed ER–endosome MCSs in a phosphatidylinositol 4-phosphate (PI4P)-dependent manner. ORP10 exhibited a lipid exchange activity toward its ligands, PI4P and phosphatidylserine (PS), between liposomes in vitro, and between the ER and endosomes in situ. Cell biological analysis demonstrated that ORP10 supplies a pool of PS from the ER, in exchange for PI4P, to endosomes where the PS-binding protein EHD1 is recruited to facilitate endosome fission. Our study highlights a novel lipid exchange at ER–endosome MCSs as a nonenzymatic PI4P-to-PS conversion mechanism that organizes membrane remodeling during retrograde membrane trafficking.     

Divergent pathways lead to ESCRT-III-catalyzed membrane fission

Endosomal sorting complexes required for transport (ESCRT) have been implicated in topologically similar but diverse cellular and pathological processes including multivesicular body (MVB) biogenesis, cytokinesis and enveloped virus budding. Although receptor sorting at the endosomal membrane producing MVBs employs the regulated assembly of ESCRT-0 followed by ESCRT-I, -II, -III and the vacuolar protein sorting (VPS)4 complex, other ESCRT-catalyzed processes require only a subset of complexes which commonly includes ESCRT-III and VPS4. Recent progress has shed light on the pathway of ESCRT assembly and highlights the separation of tasks of different ESCRT complexes and associated partners. The emerging picture suggests that among all ESCRT-catalyzed processes, divergent pathways lead to ESCRT-III assembly within the neck of a budding structure catalyzing membrane fission.     

Mitochondrial porin regulates the sensitivity of anion carriers to inhibitors

In mitoplasts, respiratory stimulation by ADP, palmitate, DNP and CCCP and sensitivity of respiration to carboxyatractylate are considerably less pronounced than in mitochondria. Addition of porin-containing preparations (purified outer membranes or solubilized mitochondrial porin) to mitoplasts results in partial restoration of the oxygen consumption and sensitivity to carboxyatractylate (CAT). The uncoupling effect of FCCP in mitoplasts is CAT-resistant and does not depend on added porin. It is suggested that mitochondrial porin may be a natural activator of ADP/ATP antiporter and succinate carrier in mitochondria.     

The mitochondrial outer membrane protein hFis1 regulates mitochondrial morphology and fission through self-interaction

Mitochondrial fission in mammals is mediated by at least two proteins, DLP1/Drp1 and hFis1. DLP1 mediates the scission of mitochondrial membranes through GTP hydrolysis, and hFis1 is a putative DLP1 receptor anchored at the mitochondrial outer membrane by a C-terminal single transmembrane domain. The cytosolic domain of hFis1 contains six α-helices (α1-α6) out of which α2-α5 form two tetratricopeptide repeat (TPR) folds. In this study, by using chimeric constructs, we demonstrated that the cytosolic domain contains the necessary information for hFis1 function during mitochondrial fission. By using transient expression of different mutant forms of the hFis1 protein, we found that hFis1 self-interaction plays an important role in mitochondrial fission. Our results show that deletion of the α1 helix greatly increased the formation of dimeric and oligomeric forms of hFis1, indicating that α1 helix functions as a negative regulator of the hFis1 self-interaction. Further mutational approaches revealed that a tyrosine residue in the α5 helix and the linker between α3 and α4 helices participate in hFis1 oligomerization. Mutations causing oligomerization defect greatly reduced the ability to induce not only mitochondrial fragmentation by full-length hFis1 but also the formation of swollen ball-shaped mitochondria caused by α1-deleted hFis1. Our data suggest that oligomerization of hFis1 in the mitochondrial outer membrane plays a role in mitochondrial fission, potentially through participating in fission factor recruitment.