Protein kinase D regulates basolateral membrane protein exit from trans-Golgi network

Protein kinase D (PKD) binds to diacylglycerol (DAG) in the trans-Golgi network (TGN) and is activated by trimeric G-protein subunits beta gamma. This complex then regulates the formation of transport carriers in the TGN that traffic to the plasma membrane in non-polarized cells. Here we report specificity of different PKD isoforms in regulating protein trafficking from the TGN. Kinase-inactive forms of PKD1, PKD2 and PKD3 localize to the TGN in polarized and non-polarized cells. PKD activity is required only for the transport of proteins containing basolateral sorting information, and seems to be cargo specific.     

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.     

Dimeric PKD regulates membrane fission to form transport carriers at the TGN

Protein kinase D (PKD) is recruited to the trans-Golgi network (TGN) through interaction with diacylglycerol (DAG) and is required for the biogenesis of TGN to cell surface transport carriers. We now provide definitive evidence that PKD has a function in membrane fission. PKD depletion by siRNA inhibits trafficking from the TGN, whereas expression of a constitutively active PKD converts TGN into small vesicles. These findings demonstrate that PKD regulates membrane fission and this activity is used to control the size of transport carriers, and to prevent uncontrolled vesiculation of TGN during protein transport.     

Regulation of leading edge microtubule and actin dynamics downstream of Rac1

Actin in migrating cells is regulated by Rho GTPases. However, Rho proteins might also affect microtubules (MTs). Here, we used time-lapse microscopy of PtK1 cells to examine MT regulation downstream of Rac1. In these cells, "pioneer" MTs growing into leading-edge protrusions exhibited a decreased catastrophe frequency and an increased time in growth as compared with MTs further from the leading edge. Constitutively active Rac1(Q61L) promoted pioneer behavior in most MTs, whereas dominant-negative Rac1(T17N) eliminated pioneer MTs, indicating that Rac1 is a regulator of MT dynamics in vivo. Rac1(Q61L) also enhanced MT turnover through stimulation of MT retrograde flow and breakage. Inhibition of p21-activated kinases (Paks), downstream effectors of Rac1, inhibited Rac1(Q61L)-induced MT growth and retrograde flow. In addition, Rac1(Q61L) promoted lamellipodial actin polymerization and Pak-dependent retrograde flow. Together, these results indicate coordinated regulation of the two cytoskeletal systems in the leading edge of migrating cells.     

A new class of carriers that transport selective cargo from the trans Golgi network to the cell surface

We have isolated a membrane fraction enriched in a class of transport carriers that form at the trans Golgi network (TGN) and are destined for the cell surface in HeLa cells. Protein kinase D (PKD) is required for the biogenesis of these carriers that contain myosin II, Rab6a, Rab8a, and synaptotagmin II, as well as a number of secretory and plasma membrane‐specific cargoes. Our findings reveal a requirement for myosin II in the migration of these transport carriers but not in their biogenesis per se. Based on the cargo secreted by these carriers we have named them CARTS for CAR riers of the T GN to the cell S urface. Surprisingly, CARTS are distinct from the carriers that transport vesicular stomatitis virus (VSV)‐G protein and collagen I from the TGN to the cell surface. Altogether, the identification of CARTS provides a valuable means to understand TGN to cell surface traffic.     

Protein Kinase D2 Assembles a Multiprotein Complex at the Trans-Golgi Network to Regulate Matrix Metalloproteinase Secretion

Vesicle formation and fission are tightly regulated at the trans-Golgi network (TGN) during constitutive secretion. Two major protein families regulate these processes: members of the adenosyl-ribosylation factor family of small G-proteins (ARFs) and the protein kinase D (PKD) family of serine/threonine kinases. The functional relationship between these two key regulators of protein transport from the TGN so far is elusive. We here demonstrate the assembly of a novel functional protein complex at the TGN and its key members: cytosolic PKD2 binds ARF-like GTPase (ARL1) and shuttles ARL1 to the TGN. ARL1, in turn, localizes Arfaptin2 to the TGN. At the TGN, where PKD2 interacts with active ARF1, PKD2, and ARL1 are required for the assembly of a complex comprising of ARF1 and Arfaptin2 leading to secretion of matrix metalloproteinase-2 and -7. In conclusion, our data indicate that PKD2 is a core factor in the formation of this multiprotein complex at the TGN that controls constitutive secretion of matrix metalloproteinase cargo.     

Post-Golgi anterograde transport requires GARP-dependent endosome-to-TGN retrograde transport

The importance of endosome-to–trans-Golgi network (TGN) retrograde transport in the anterograde transport of proteins is unclear. In this study, genome-wide screening of the factors necessary for efficient anterograde protein transport in human haploid cells identified subunits of the Golgi-associated retrograde protein (GARP) complex, a tethering factor involved in endosome-to-TGN transport. Knockout (KO) of each of the four GARP subunits, VPS51–VPS54, in HEK293 cells caused severely defective anterograde transport of both glycosylphosphatidylinositol (GPI)-anchored and transmembrane proteins from the TGN. Overexpression of VAMP4, v-SNARE, in VPS54-KO cells partially restored not only endosome-to-TGN retrograde transport, but also anterograde transport of both GPI-anchored and transmembrane proteins. Further screening for genes whose overexpression normalized the VPS54-KO phenotype identified TMEM87A, encoding an uncharacterized Golgi-resident membrane protein. Overexpression of TMEM87A or its close homologue TMEM87B in VPS54-KO cells partially restored endosome-to-TGN retrograde transport and anterograde transport. Therefore GARP- and VAMP4-dependent endosome-to-TGN retrograde transport is required for recycling of molecules critical for efficient post-Golgi anterograde transport of cell-surface integral membrane proteins. In addition, TMEM87A and TMEM87B are involved in endosome-to-TGN retrograde transport.     

PKCeta is required for beta1gamma2/beta3gamma2- and PKD-mediated transport to the cell surface and the organization of the Golgi apparatus

Protein kinase D (PKD) binds to a pool of diacylglycerol (DAG) in the TGN and undergoes a process of activation that involves heterotrimeric GTP-binding protein subunits betagamma to regulate membrane fission. This fission reaction is used to generate transport carriers at the TGN that are en route to the cell surface. We now report that PKD is activated specifically by G protein subunit beta1gamma2 and beta3gamma2 via the Golgi apparatus-associated PKCeta. Compromising the kinase activity of PKCeta-inhibited protein transport from TGN to the cell surface. Expression of constitutively activated PKCeta caused Golgi fragmentation, which was inhibited by a kinase inactive form of PKD. Our findings reveal that betagamma, PKCeta, and PKD act in series to generate transport carriers from the TGN and their overactivation results in complete vesiculation of the Golgi apparatus.     

Regulation of secretory transport by protein kinase D-mediated phosphorylation of the ceramide transfer protein

Protein kinase D (PKD) has been identified as a crucial regulator of secretory transport at the trans-Golgi network (TGN). Recruitment and activation of PKD at the TGN is mediated by the lipid diacylglycerol, a pool of which is generated by sphingomyelin synthase from ceramide and phosphatidylcholine. The nonvesicular transfer of ceramide from the endoplasmic reticulum to the Golgi complex is mediated by the lipid transfer protein CERT (ceramide transport). In this study, we identify CERT as a novel in vivo PKD substrate. Phosphorylation on serine 132 by PKD decreases the affinity of CERT toward its lipid target phosphatidylinositol 4-phosphate at Golgi membranes and reduces ceramide transfer activity, identifying PKD as a regulator of lipid homeostasis. We also show that CERT, in turn, is critical for PKD activation and PKD-dependent protein cargo transport to the plasma membrane. Thus, the interdependence of PKD and CERT is key to the maintenance of Golgi membrane integrity and secretory transport.     

Arl5b is a Golgi-localised small G protein involved in the regulation of retrograde transport

Regulation of membrane transport is controlled by small G proteins, which include members of the Rab and Arf families. Whereas the role of the classic Arf family members are well characterized, many of the Arf-like proteins (Arls) remain poorly defined. Here we show that Arl5a and Arl5b are localised to the trans-Golgi in mammalian cells, and furthermore have identified a role for Arl5b in the regulation of retrograde membrane transport from endosomes to the trans-Golgi network (TGN). The constitutively active Arl5b (Q70L)-GFP mutant was localised efficiently to the Golgi in HeLa cells whereas the dominant-negative Arl5b (T30N)-GFP mutant was dispersed throughout the cytoplasm and resulted in perturbation of the Golgi apparatus. Stable HeLa cells expressing GFP-tagged Arl5b (Q70L) showed an increased rate of endosome-to-Golgi transport of the membrane cargo TGN38 compared with control HeLa cells. Depletion of Arl5b by RNAi resulted in an alteration in the intracellular distribution of mannose-6-phosphate receptor, and significantly reduced the endosome-to-TGN transport of the membrane cargo TGN38 and of Shiga toxin, but had no affect on the anterograde transport of the cargo E-cadherin. Collectively these results suggest that Arl5b is a TGN-localised small G protein that plays a key role in regulating transport along the endosome-TGN pathway.     

Memo-RhoA-mDia1 signaling controls microtubules, the actin network, and adhesion site formation in migrating cells

Actin assembly at the cell front drives membrane protrusion and initiates the cell migration cycle. Microtubules (MTs) extend within forward protrusions to sustain cell polarity and promote adhesion site turnover. Memo is an effector of the ErbB2 receptor tyrosine kinase involved in breast carcinoma cell migration. However, its mechanism of action remained unknown. We report in this study that Memo controls ErbB2-regulated MT dynamics by altering the transition frequency between MT growth and shortening phases. Moreover, although Memo-depleted cells can assemble the Rac1-dependent actin meshwork and form lamellipodia, they show defective localization of lamellipodial markers such as α-actinin-1 and a reduced number of short-lived adhesion sites underlying the advancing edge of migrating cells. Finally, we demonstrate that Memo is required for the localization of the RhoA guanosine triphosphatase and its effector mDia1 to the plasma membrane and that Memo–RhoA–mDia1 signaling coordinates the organization of the lamellipodial actin network, adhesion site formation, and MT outgrowth within the cell leading edge to sustain cell motility.     

A novel mechanism for localizing membrane proteins to yeast trans-Golgi network requires function of synaptojanin-like protein

Localization of resident membrane proteins to the yeast trans-Golgi network (TGN) involves both their retrieval from a prevacuolar/endosomal compartment (PVC) and a "slow delivery" mechanism that inhibits their TGN-to-PVC transport. A screen for genes required for the slow delivery mechanism uncovered INP53, a gene encoding a phosphoinositide phosphatase. A retrieval-defective model TGN protein, A(F-->A)-ALP, was transported to the vacuole in inp53 mutants approximately threefold faster than in wild type. Inp53p appears to function in a process distinct from PVC retrieval because combining inp53 with mutations that block retrieval resulted in a much stronger phenotype than either mutation alone. In vps27 strains defective for both anterograde and retrograde transport out of the PVC, a loss of Inp53p function markedly accelerated the rate of transport of TGN residents A-ALP and Kex2p into the PVC. Inp53p function is cargo specific because a loss of Inp53p function had no effect on the rate of Vps10p transport to the PVC in vps27 cells. The rate of early secretory pathway transport appeared to be unaffected in inp53 mutants. Cell fractionation experiments suggested that Inp53p associates with Golgi or endosomal membranes. Taken together, these results suggest that a phosphoinositide signaling event regulates TGN-to-PVC transport of select cargo proteins.     

Endosome-to-Golgi transport pathways in physiological processes

The trans-Golgi network (TGN) is a major traffic hub of the cell, as it regulates membrane transport in the secretory pathway as well as receiving protein cargo by retrograde transport from endocytic compartments. Retrograde transport between endosomes and the TGN is essential for the recycling of membrane proteins which regulate a range of cellular and development functions. In addition, retrograde transport pathways are exploited by many bacterial toxins to mediate cytotoxicity and by some viral proteins to promote pathogenicity. Recent advances using a range of molecular cell biological strategies have identified multiple retrograde transport pathways each regulated by a distinct set of molecular machinery. Here we review recent advances in this field and highlight the importance of these transport pathways in a range of physiological processes.     

Roles of type II myosin and a tropomyosin isoform in retrograde actin flow in budding yeast

Retrograde flow of cortical actin networks and bundles is essential for cell motility and retrograde intracellular movement, and for the formation and maintenance of microvilli, stereocilia, and filopodia. Actin cables, which are F-actin bundles that serve as tracks for anterograde and retrograde cargo movement in budding yeast, undergo retrograde flow that is driven, in part, by actin polymerization and assembly. We find that the actin cable retrograde flow rate is reduced by deletion or delocalization of the type II myosin Myo1p, and by deletion or conditional mutation of the Myo1p motor domain. Deletion of the tropomyosin isoform Tpm2p, but not the Tpm1p isoform, increases the rate of actin cable retrograde flow. Pretreatment of F-actin with Tpm2p, but not Tpm1p, inhibits Myo1p binding to F-actin and Myo1p-dependent F-actin gliding. These data support novel, opposing roles of Myo1p and Tpm2 in regulating retrograde actin flow in budding yeast and an isoform-specific function of Tpm1p in promoting actin cable function in myosin-driven anterograde cargo transport.     

Fibroblast growth factor-2 and remodeled type I collagen control membrane protrusion in human vascular smooth muscle cells: biphasic activation of Rac1

Plasma membrane protrusion is fundamental to cell motility, but its regulation by the extracellular environment is not well elucidated. We have quantified lamellipodial protrusion dynamics in human vascular smooth muscle cells exposed to fibroblast growth factor 2 (FGF-2) and type I collagen, two distinct ligands presented to vascular cells during arterial remodeling. Video microscopy revealed that FGF-2 stimulated a modest increase in lamellipodial protrusion rate that peaked within 15 min. This response was associated with immediate but transient activation of Rac1 and was inhibited in cells infected with retrovirus containing cDNA encoding dominant-negative Rac1. A 1-h exposure to FGF-2 also set up a second phase of more striking lamellipodial protrusion evident at 24-36 h. This delayed response was most pronounced when cells were on type 1 collagen and was associated with FGF-2-induced expression of collagenase-1 that localized to the edge of protruding lamellipodia. Moreover, late membrane protrusion was inhibited when cells were on collagenase-resistant type I collagen, implicating degraded collagen as a mediator. For cells on collagen, the immediate activation of Rac1 by FGF-2 was followed by a sustained wave of Rac1 activation that was inhibited when cleavage of the collagen triple helix was prevented and also by blockade of alpha(v)beta(3) integrin. We conclude that lamellipodial protrusion in smooth muscle cells can be regulated by waves of Rac1 activation, corresponding to the sequential presentation of FGF-2 and remodeled collagen. The findings thus reveal a previously unrecognized level of coordination among extracellular input that enables cells to maintain protrusive activity over prolonged periods.     

The golgin GCC88 is required for efficient retrograde transport of cargo from the early endosomes to the trans-Golgi network

Retrograde transport pathways from early/recycling endosomes to the trans-Golgi network (TGN) are poorly defined. We have investigated the role of TGN golgins in retrograde trafficking. Of the four TGN golgins, p230/golgin-245, golgin-97, GCC185, and GCC88, we show that GCC88 defines a retrograde transport pathway from early endosomes to the TGN. Depletion of GCC88 in HeLa cells by interference RNA resulted in a block in plasma membrane-TGN recycling of two cargo proteins, TGN38 and a CD8 mannose-6-phosphate receptor cytoplasmic tail fusion protein. In GCC88-depleted cells, cargo recycling was blocked in the early endosome. Depletion of GCC88 dramatically altered the TGN localization of the t-SNARE syntaxin 6, a syntaxin required for endosome to TGN transport. Furthermore, the transport block in GCC88-depleted cells was rescued by syntaxin 6 overexpression. Internalized Shiga toxin was efficiently transported from endosomes to the Golgi of GCC88-depleted cells, indicating that Shiga toxin and TGN38 are internalized by distinct retrograde transport pathways. These findings have identified an essential role for GCC88 in the localization of TGN fusion machinery for transport from early endosomes to the TGN, and they have allowed the identification of a retrograde pathway which differentially selects TGN38 and mannose-6-phosphate receptor from Shiga toxin.     

Segregation of COPI-rich and anterograde-cargo-rich domains in endoplasmic-reticulum-to-Golgi transport complexes.

Membrane traffic between the endoplasmic reticulum (ER) and the Golgi complex is regulated by two vesicular coat complexes, COPII and COPI. COPII has been implicated in the selective packaging of anterograde cargo into coated transport vesicles budding from the ER [1]. In mammalian cells, these vesicles coalesce to form tubulo-vesicular transport complexes (TCs), which shuttle anterograde cargo from the ER to the Golgi complex [2] [3] [4]. In contrast, COPI-coated vesicles are proposed to mediate recycling of proteins from the Golgi complex to the ER [1] [5] [6] [7]. The binding of COPI to COPII-coated TCs [3] [8] [9], however, has led to the proposal that COPI binds to TCs and specifically packages recycling proteins into retrograde vesicles for return to the ER [3] [9]. To test this hypothesis, we tracked fluorescently tagged COPI and anterograde-transport markers simultaneously in living cells. COPI predominated on TCs shuttling anterograde cargo to the Golgi complex and was rarely observed on structures moving in directions consistent with retrograde transport. Furthermore, a progressive segregation of COPI-rich domains and anterograde-cargo-rich domains was observed in the TCs. This segregation and the directed motility of COPI-containing TCs were inhibited by antibodies that blocked COPI function. These observations, which are consistent with previous biochemical data [2] [9], suggest a role for COPI within TCs en route to the Golgi complex. By sequestering retrograde cargo in the anterograde-directed TCs, COPI couples the sorting of ER recycling proteins [10] to the transport of anterograde cargo.     

Protein kinase D: activation for Golgi carrier formation

Protein kinase D regulates fission at the trans-Golgi network (TGN) of transport carriers that deliver cargo to the plasma membrane. PKD is first recruited to the TGN through interaction with diacylglycerol and is subsequently activated by phosphorylation to promote carrier fission. In a recent study, the relevant upstream kinase at the TGN was identified as the novel protein kinase C isoform PKCeta, which in turn is activated in response to heterotrimeric G-protein activation. These findings indicate the existence of a kinase signaling cascade at the TGN that regulates carrier fission and suggest a mechanism by which cargo might direct the formation of its transport carriers.     

A Highlights from MBoC Selection: Insulin-promoted mobilization of GLUT4 from a perinuclear storage site requires RAB10

Insulin controls glucose uptake into muscle and fat cells by inducing a net redistribution of glucose transporter 4 (GLUT4) from intracellular storage to the plasma membrane (PM). The TBC1D4-RAB10 signaling module is required for insulin-stimulated GLUT4 translocation to the PM, although where it intersects GLUT4 traffic was unknown. Here we demonstrate that TBC1D4-RAB10 functions to control GLUT4 mobilization from a trans-Golgi network (TGN) storage compartment, establishing that insulin, in addition to regulating the PM proximal effects of GLUT4-containing vesicles docking to and fusion with the PM, also directly regulates the behavior of GLUT4 deeper within the cell. We also show that GLUT4 is retained in an element/domain of the TGN from which newly synthesized lysosomal proteins are targeted to the late endosomes and the ATP7A copper transporter is translocated to the PM by elevated copper. Insulin does not mobilize ATP7A nor does copper mobilize GLUT4, and RAB10 is not required for copper-elicited ATP7A mobilization. Consequently, GLUT4 intracellular sequestration and mobilization by insulin is achieved, in part, through utilizing a region of the TGN devoted to specialized cargo transport in general rather than being specific for GLUT4. Our results define the GLUT4-containing region of the TGN as a sorting and storage site from which different cargo are mobilized by distinct signals through unique molecular machinery.     

Role of the Second Cysteine-rich Domain and Pro275 in Protein Kinase D2 Interaction with ADP-Ribosylation Factor 1, Trans-Golgi Network Recruitment,and Protein Transport

Protein kinase D (PKD) isoenzymes regulate the formation of transport carriers from the trans-Golgi network (TGN) that are en route to the plasma membrane. The PKD C1a domain is required for the localization of PKDs at the TGN. However, the precise mechanism of how PKDs are recruited to the TGN is still elusive. Here, we report that ADP-ribosylation factor (ARF1), a small GTPase of the Ras superfamily and a key regulator of secretory traffic, specifically interacts with PKD isoenzymes. ARF1, but not ARF6, binds directly to the second cysteine-rich domain (C1b) of PKD2, and precisely to Pro275 within this domain. Pro275 in PKD2 is not only crucial for the PKD2-ARF1 interaction but also for PKD2 recruitment to and PKD2 function at the TGN, namely, protein transport to the plasma membrane. Our data suggest a novel model in which ARF1 recruits PKD2 to the TGN by binding to Pro275 in its C1b domain followed by anchoring of PKD2 in the TGN membranes via binding of its C1a domain to diacylglycerol. Both processes are critical for PKD2-mediated protein transport.