Conserved oligomeric Golgi complex specifically regulates the maintenance of Golgi glycosylation machinery

Cell surface lectin staining, examination of Golgi glycosyltransferases stability and localization, and matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) analysis were employed to investigate conserved oligomeric Golgi (COG)-dependent glycosylation defects in HeLa cells. Both Griffonia simplicifolia lectin-II and Galanthus nivalus lectins were specifically bound to the plasma membrane glycoconjugates of COG-depleted cells, indicating defects in activity of medial- and trans-Golgi-localized enzymes. In response to siRNA-induced depletion of COG complex subunits, several key components of Golgi glycosylation machinery, including MAN2A1, MGAT1, B4GALT1 and ST6GAL1, were severely mislocalized. MALDI-TOF analysis of total N-linked glycoconjugates indicated a decrease in the relative amount of sialylated glycans in both COG3 KD and COG4 KD cells. In agreement to a proposed role of the COG complex in retrograde membrane trafficking, all types of COG-depleted HeLa cells were deficient in the Brefeldin A- and Sar1 DN-induced redistribution of Golgi resident glycosyltransferases to the endoplasmic reticulum. The retrograde trafficking of medial- and trans-Golgi-localized glycosylation enzymes was affected to a larger extent, strongly indicating that the COG complex regulates the intra-Golgi protein movement. COG complex-deficient cells were not defective in Golgi re-assembly after the Brefeldin A washout, confirming specificity in the retrograde trafficking block. The lobe B COG subcomplex subunits COG6 and COG8 were localized on trafficking intermediates that carry Golgi glycosyltransferases, indicating that the COG complex is directly involved in trafficking and maintenance of Golgi glycosylation machinery.     

Actin and Arf1-dependent recruitment of a cortactin-dynamin complex to the Golgi regulates post-Golgi transport

Cortactin is an actin-binding protein that has recently been implicated in endocytosis. It binds directly to dynamin-2 (Dyn2), a large GTPase that mediates the formation of vesicles from the plasma membrane and the Golgi. Here we show that cortactin associates with the Golgi to regulate the actin- and Dyn2-dependent transport of cargo. Cortactin antibodies stain the Golgi apparatus, labelling peripheral buds and vesicles that are associated with the cisternae. Notably, in vitro or intact-cell experiments show that activation of Arf1 mediates the recruitment of actin, cortactin and Dyn2 to Golgi membranes. Furthermore, selective disruption of the cortactin-Dyn2 interaction significantly reduces the levels of Dyn2 at the Golgi and blocks the transit of nascent proteins from the trans-Golgi network, resulting in swollen and distended cisternae. These findings support the idea of an Arf1-activated recruitment of an actin, cortactin and Dyn2 complex that is essential for Golgi function.     

Lysophosphatidic acid acyltransferase 3 regulates Golgi complex structure and function

Recent studies have suggested that the functional organization of the Golgi complex is dependent on phospholipid remodeling enzymes. Here, we report the identification of an integral membrane lysophosphatidic acid–specific acyltransferase, LPAAT3, which regulates Golgi membrane tubule formation, trafficking, and structure by altering phospholipids and lysophospholipids. Overexpression of LPAAT3 significantly inhibited the formation of Golgi membrane tubules in vivo and in vitro. Anterograde and retrograde protein trafficking was slower in cells overexpressing LPAAT3 and accelerated in cells with reduced expression (by siRNA). Golgi morphology was also dependent on LPAAT3 because its knockdown caused the Golgi to become fragmented. These data are the first to show a direct role for a specific phospholipid acyltransferase in regulating membrane trafficking and organelle structure.     

B2-1, a Sec7- and pleckstrin homology domain-containing protein, localizes to the Golgi complex

B2-1 is a human protein that contains both a Sec7 and a pleckstrin homology domain. The yeast Sec7 protein was previously shown to be involved in vesicle formation in the Golgi and endoplasmic reticulum. Recently, several groups have shown that B2-1 and highly similar proteins (e.g., ARNO, ARNO3) have varied cellular functions and subcellular locations. One of these is an association of the B2-1 Sec7 domain with the plasma membrane, binding to the cytoplasmic portion of the integrin beta2 chain (CD18) and is postulated to be involved in inside-out signaling. Other groups have shown that B2-1 and these related proteins are guanine nucleotide-exchange factors that act upon ADP ribosylation factors (ARFs) and are localized to the Golgi or plasma membrane. Here we report the subcellular localization of B2-1 protein. Interestingly, B2-1 does not localize to the plasma membrane but rather associates with a distinct Golgi complex compartment. B2-1's distribution can be disrupted by brefeldin A, a drug that rapidly disrupts the Golgi apparatus by inhibiting ARF activity. Furthermore, transient transfection of GFP-tagged B2-1 shows Golgi complex targeting. Excessive overexpression of transfected B2-1 causes partial Golgi dispersion.     

ERK1c regulates Golgi fragmentation during mitosis

Extracellular signal-regulated kinase 1c (ERK1c) is an alternatively spliced form of ERK1 that is regulated differently than other ERK isoforms. We studied the Golgi functions of ERK1c and found that it plays a role in MEK-induced mitotic Golgi fragmentation. Thus, in late G2 and mitosis of synchronized cells, the expression and activity of ERK1c was increased and it colocalized mainly with Golgi markers. Small interfering RNA of ERK1c significantly attenuated, whereas ERK1c overexpression facilitated, mitotic Golgi fragmentation. These effects were also reflected in mitotic progression, indicating that ERK1c is involved in cell cycle regulation via modulation of Golgi fragmentation. Although ERK1 was activated in mitosis as well, it could not replace ERK1c in regulating Golgi fragmentation. Therefore, MEKs regulate mitosis via all three ERK isoforms, where ERK1c acts specifically in the Golgi, whereas ERK1 and 2 regulate other mitosis-related processes. Thus, ERK1c extends the specificity of the Ras-MEK cascade by activating ERK1/2-independent processes.     

The cyclin A1-CDK2 complex regulates DNA double-strand break repair

Vertebrates express two A-type cyclins; both associate with and activate the CDK2 protein kinase. Cyclin A1 is required in the male germ line, but its molecular functions are incompletely understood. We observed specific induction of cyclin A1 expression and promoter activity after UV and gamma-irradiation which was mediated by p53. cyclin A1-/- cells showed increased radiosensitivity. To unravel a potential role of cyclin A1 in DNA repair, we performed a yeast triple hybrid screen and identified the Ku70 DNA repair protein as a binding partner and substrate of the cyclin A1-CDK2 complex. DNA double-strand break (DSB) repair was deficient in cyclin A1-/- cells. Further experiments indicated that A-type cyclins activate DNA DSB repair by mechanisms that depend on CDK2 activity and Ku proteins. Both cyclin A1 and cyclin A2 enhanced DSB repair by homologous recombination, but only cyclin A1 significantly activated nonhomologous end joining. DNA DSB repair was specific for A-type cyclins because cyclin E was ineffective. These findings establish a novel function for cyclin A1 and CDK2 in DNA DSB repair following radiation damage.     

The Rab6 GTPase regulates recruitment of the dynactin complex to Golgi membranes

Dynactin is a multisubunit protein complex required for the activity of dynein in diverse intracellular motility processes, including membrane transport. Dynactin can bind to vesicles and liposomes containing acidic phospholipids, but general properties such as this are unlikely to explain the regulated recruitment of dynactin to specific sites on organelle membranes. Additional factors must therefore exist to control this process. Candidates for these factors are the Rab GTPases, which function in the tethering of vesicles to their target organelle prior to membrane fusion. In particular, Rab27a tethers melanosomes to the actin cytoskeleton. Other Rabs have been implicated in microtubule-dependent organelle motility; Rab7 controls lysosomal transport, and Rab6 is involved in microtubule-dependent transport pathways through the Golgi and from endosomes to the Golgi. We demonstrate that dynactin binds to Rab6 and shows a Rab6-dependent recruitment to Golgi membranes. Other Golgi Rabs do not bind to dynactin and are unable to support its recruitment to membranes. Rab6 therefore functions as a specificity or tethering factor controlling the recruitment of dynactin to membranes.     

Multiple activities for Arf1 at the Golgi complex

The Arf family of GTPases regulates membrane traffic and organelle structure. At the Golgi complex, Arf proteins facilitate membrane recruitment of many cytoplasmic coat proteins to allow sorting of membrane proteins for transport, stimulate the activity of enzymes that modulate the lipid composition of the Golgi, and assemble a cytoskeletal scaffold on the Golgi. Arf1 is the Arf family member most closely studied for its function at the Golgi complex. A number of regulators that activate and inactivate Arf1 on the Golgi have been described that localize to different regions of the organelle. This spatial distribution of Arf regulators may facilitate the recruitment of the coat proteins and other Arf effectors to different regions of the Golgi complex.     

Exiting the Golgi complex

The composition and identity of cell organelles are dictated by the flux of lipids and proteins that they receive and lose through cytosolic exchange and membrane trafficking. The trans-Golgi network (TGN) is a major sorting centre for cell lipids and proteins at the crossroads of the endocytic and exocytic pathways; it has a complex dynamic structure composed of a network of tubular membranes that generate pleiomorphic carriers targeted to different destinations. Live-cell imaging combined with three-dimensional tomography has recently provided the temporal and topographical framework that allows the assembly of the numerous molecular machineries so far implicated in sorting and trafficking at the TGN.     

A PACS-1, GGA3 and CK2 complex regulates CI-MPR trafficking

The cation-independent mannose-6-phosphate receptor (CI-MPR) follows a highly regulated sorting itinerary to deliver hydrolases from the trans-Golgi network (TGN) to lysosomes. Cycling of CI-MPR between the TGN and early endosomes is mediated by GGA3, which directs TGN export, and PACS-1, which directs endosome-to-TGN retrieval. Despite executing opposing sorting steps, GGA3 and PACS-1 bind to an overlapping CI-MPR trafficking motif and their sorting activity is controlled by the CK2 phosphorylation of their respective autoregulatory domains. However, how CK2 coordinates these opposing roles is unknown. We report a CK2-activated phosphorylation cascade controlling PACS-1- and GGA3-mediated CI-MPR sorting. PACS-1 links GGA3 to CK2, forming a multimeric complex required for CI-MPR sorting. PACS-1-bound CK2 stimulates GGA3 phosphorylation, releasing GGA3 from CI-MPR and early endosomes. Bound CK2 also phosphorylates PACS-1Ser(278), promoting binding of PACS-1 to CI-MPR to retrieve the receptor to the TGN. Our results identify a CK2-controlled cascade regulating hydrolase trafficking and sorting of itinerant proteins in the TGN/endosomal system.     

A large-conductance anion channel of the Golgi complex

An acidic lumenal pH is vital for the proper posttranslational modifications and sorting of proteins and lipids from the Golgi complex. We characterized ion channels present in Golgi fractions that have been cleared of transiting proteins. A large conductance anion channel was observed in approximately 30% of successful channel incorporations into the planar lipid bilayer. The channel, GOLAC-2, has six levels (one closed and five open). The open states are each approximately 20% increments of the maximal, 325 pS conductance. The channel was approximately 6 times more selective for Cl(-) over K(+). Binomial analysis of percent occupancy for each conducting level supports the hypothesis of five independent conducting pathways. The conducting levels can coordinately gate because full openings and closings were often observed. Addition of 3 to 5 mM reduced glutathione to the cis chamber caused dose-dependent increases in single channel conductance, indicating that the channel may be regulated by the oxidation-reduction state of the cell. We propose that GOLAC-2 is a co-channel complex consisting of five identical pores that have a coordinated gating mechanism. GOALC-2 may function as a source of counter anions for the H(+)-ATPase and may be involved in regulating charge balance and membrane potential of the Golgi complex.     

A role for kinesin-2 in COPI-dependent recycling between the ER and the Golgi complex

Transport carriers operating between early compartments in the mammalian secretory pathway have to travel long distances in the cell by mostly relying on the microtubule network and its associated motor proteins. Although anterograde transport from the endoplasmic reticulum (ER) to the Golgi complex is mediated by cytoplasmic dynein, the identity of the motor(s) mediating transport in the retrograde direction is presently unclear. Some studies have suggested that the heterotrimeric kinesin-2 complex plays a role in transport between the ER and the Golgi. Here, we have examined kinesin-2 function by using an RNA-interference approach to downregulate the expression of KAP3, the nonmotor subunit of kinesin-2, in HeLa cells. KAP3 silencing results in the fragmentation of the Golgi apparatus and a change in the steady-state localization of the KDEL-receptor (KDEL-R). Using specific transport assays, we show that the rate of anterograde secretory traffic is unaffected in these cells but that KDEL-R-dependent retrograde transport is strongly abrogated. Our data strongly support a role for kinesin-2 in the KDEL-R-/COPI-dependent retrograde transport pathway from the Golgi complex to the ER.     

The rubella virus E2 and E1 spike glycoproteins are targeted to the Golgi complex

Rubella virus (RV) has been reported to bud from intracellular membranes in certain cell types. In this study the intracellular site of targeting of RV envelope E2 and E1 glycoproteins has been investigated in three different cell types (CHO, BHK-21 and Vero cells) transfected with a cDNA encoding the two glycoproteins. By indirect immunofluorescence, E2 and E1 were localized to the Golgi region of all three cell types, and their distribution was disrupted by treatment with BFA or nocodazole. Immunogold labeling demonstrated that E2 and E1 were localized to Golgi cisternae and indicated that the glycoproteins were distributed across the Golgi stack. Analysis of immunoprecipitates obtained from stably transfected CHO cells revealed that E2 and E1 become endo H resistant and undergo sialylation without being transported to the cell surface. Transport of RV glycoproteins to the Golgi complex was relatively slow (t1/2 = 60-90 min). Coprecipitation experiments indicated that E2 and E1 form a heterodimer in the RER. E1 was found to fold much more slowly than E2, suggesting that the delay in transport of the heterodimer to the Golgi may be due to the slow maturation of E1 in the ER. These results indicate that RV glycoproteins behave as integral membrane proteins of the Golgi complex and thus provide a useful model to study targeting and turnover of type I membrane proteins in this organelle.     

The Par polarity complex regulates Rap1- and chemokine-induced T cell polarization

Cell polarization is required for virtually all functions of T cells, including transendothelial migration in response to chemokines. However, the molecular pathways that establish T cell polarity are poorly understood. We show that the activation of the partitioning defective (Par) polarity complex is a key event during Rap1- and chemokine-induced T cell polarization. Intracellular localization and activation of the Par complex are initiated by Rap1 and require Cdc42 activity. The Rac activator Tiam1 associates with both Rap1 and components of the Par complex, and thereby may function to connect the Par polarity complex to Rap1 and to regulate the Rac-mediated actin remodelling required for T cell polarization. Consistent with these findings, Tiam1-deficient T cells are impaired in Rap1- and chemokine-induced polarization and chemotaxis. Our studies implicate Tiam1 and the Par polarity complex in polarization of T cells, and provide a mechanism by which chemokines and Rap1 regulate T cell polarization and chemotaxis.