The Kinetics of Mannose 6-Phosphate Receptor Trafficking in the Endocytic Pathway in HEp-2 Cells: The Receptor Enters and Rapidly Leaves Multivesicular Endosomes without Accumulating in a Prelysosomal Compartment

We have previously shown that in HEp-2 cells, multivesicular bodies (MVBs) processing internalized epidermal growth factor–epidermal growth factor receptor complexes mature and fuse directly with lysosomes in which the complexes are degraded. The MVBs do not fuse with a prelysosomal compartment enriched in mannose 6-phosphate receptor (M6PR) as has been described in other cell types. Here we show that the cation-independent M6PR does not become enriched in the endocytic pathway en route to the lysosome, but if a pulse of M6PR or an M6PR ligand, cathepsin D, is followed, a significant fraction of these proteins are routed from the trans-Golgi to MVBs. Accumulation of M6PR does not occur because when the ligand dissociates, the receptor rapidly leaves the MVB. At steady state, most M6PR are distributed within the trans-Golgi and trans-Golgi network and in vacuolar structures distributed in the peripheral cytoplasm. We suggest that these M6PR-rich vacuoles are on the return route from MVBs to the trans-Golgi network and that a separate stable M6PR-rich compartment equivalent to the late endosome/prelysosome stage does not exist on the endosome–lysosome pathway in these cells.

Mechanisms Governing the Activation and Trafficking of Yeast G Protein-coupled Receptors

We have addressed the mechanisms governing the activation and trafficking of G protein-coupled receptors (GPCRs) by analyzing constitutively active mating pheromone receptors (Ste2p and Ste3p) of the yeast Saccharomyces cerevisiae. Substitution of the highly conserved proline residue in transmembrane segment VI of these receptors causes constitutive signaling. This proline residue may facilitate folding of GPCRs into native, inactive conformations, and/or mediate agonist-induced structural changes leading to G protein activation. Constitutive signaling by mutant receptors is suppressed upon coexpression with wild-type, but not G protein coupling-defective, receptors. Wild-type receptors may therefore sequester a limiting pool of G proteins; this apparent “precoupling” of receptors and G proteins could facilitate signal production at sites where cell surface projections form during mating partner discrimination. Finally, rather than being expressed mainly at the cell surface, constitutively active pheromone receptors accumulate in post-endoplasmic reticulum compartments. This is in contrast to other defective membrane proteins, which apparently are targeted by default to the vacuole. We suggest that the quality-control mechanism that retains receptors in post-endoplasmic reticulum compartments may normally allow wild-type receptors to fold into their native, fully inactive conformations before reaching the cell surface. This may ensure that receptors do not trigger a response in the absence of agonist.

Dimerization of the Polymeric Immunoglobulin Receptor Controls Its Transcytotic Trafficking

Binding of dimeric immunoglobulin (Ig)A to the polymeric Ig receptor (pIgR) stimulates transcytosis of pIgR across epithelial cells. Through the generation of a series of pIgR chimeric constructs, we have tested the ability of ligand to promote receptor dimerization and the subsequent role of receptor dimerization on its intracellular trafficking. Using the cytoplasmic domain of the T cell receptor-ζ chain as a sensitive indicator of receptor oligomerization, we show that a pIgR:ζ chimeric receptor expressed in Jurkat cells initiates a ζ-specific signal transduction cascade when exposed to dimeric or tetrameric IgA, but not when exposed to monomeric IgA. In addition, we replaced the pIgR’s transmembrane domain with that of glycophorin A to force dimerization or with a mutant glycophorin transmembrane domain to prevent dimerization. Forcing dimerization stimulated transcytosis of the chimera, whereas preventing dimerization abolished ligand-stimulated transcytosis. We conclude that binding of dimeric IgA to the pIgR induces its dimerization and that this dimerization is necessary and sufficient to stimulate pIgR transcytosis.

Phosphatidylinositol 3-Kinase-mediated Endocytosis of Renal Na+,K+-ATPase α Subunit in Response to Dopamine

Dopamine (DA) inhibition of Na+,K+-ATPase in proximal tubule cells is associated with increased endocytosis of its α and β subunits into early and late endosomes via a clathrin vesicle-dependent pathway. In this report we evaluated intracellular signals that could trigger this mechanism, specifically the role of phosphatidylinositol 3-kinase (PI 3-K), the activation of which initiates vesicular trafficking and targeting of proteins to specific cell compartments. DA stimulated PI 3-K activity in a time- and dose-dependent manner, and this effect was markedly blunted by wortmannin and LY 294002. Endocytosis of the Na+,K+-ATPase α subunit in response to DA was also inhibited in dose-dependent manner by wortmannin and LY 294002. Activation of PI 3-K generally occurs by association with tyrosine kinase receptors. However, in this study immunoprecipitation with a phosphotyrosine antibody did not reveal PI 3-K activity. DA-stimulated endocytosis of Na+,K+-ATPase α subunits required protein kinase C, and the ability of DA to stimulate PI 3-K was blocked by specific protein kinase C inhibitors. Activation of PI 3-K is mediated via the D1 receptor subtype and the sequential activation of phospholipase A2, arachidonic acid, and protein kinase C. The results indicate a key role for activation of PI 3-K in the endocytic sequence that leads to internalization of Na+,K+-ATPase α subunits in response to DA, and suggest a mechanism for the participation of protein kinase C in this process.

Immunoisolation and Characterization of a Subdomain of the Endoplasmic Reticulum That Concentrates Proteins Involved in COPII Vesicle Biogenesis

Rubella virus E1 glycoprotein normally complexes with E2 in the endoplasmic reticulum (ER) to form a heterodimer that is transported to and retained in the Golgi complex. In a previous study, we showed that in the absence of E2, unassembled E1 subunits accumulate in a tubular pre-Golgi compartment whose morphology and biochemical properties are distinct from both rough ER and Golgi. We hypothesized that this compartment corresponds to hypertrophied ER exit sites that have expanded in response to overexpression of E1. In the present study we constructed BHK cells stably expressing E1 protein containing a cytoplasmically disposed epitope and isolated the pre-Golgi compartment from these cells by cell fractionation and immunoisolation. Double label indirect immunofluorescence in cells and immunoblotting of immunoisolated tubular networks revealed that proteins involved in formation of ER-derived transport vesicles, namely p58/ERGIC 53, Sec23p, and Sec13p, were concentrated in the E1-containing pre-Golgi compartment. Furthermore, budding structures were evident in these membrane profiles, and a highly abundant but unknown 65-kDa protein was also present. By comparison, marker proteins of the rough ER, Golgi, and COPI vesicles were not enriched in these membranes. These results demonstrate that the composition of the tubular networks corresponds to that expected of ER exit sites. Accordingly, we propose the name SEREC (smooth ER exit compartment) for this structure.

A Novel Tetanus Neurotoxin-insensitive Vesicle-associated Membrane Protein in SNARE Complexes of the Apical Plasma Membrane of Epithelial Cells

The importance of soluble N-ethyl maleimide (NEM)-sensitive fusion protein (NSF) attachment protein (SNAP) receptors (SNAREs) in synaptic vesicle exocytosis is well established because it has been demonstrated that clostridial neurotoxins (NTs) proteolyze the vesicle SNAREs (v-SNAREs) vesicle-associated membrane protein (VAMP)/brevins and their partners, the target SNAREs (t-SNAREs) syntaxin 1 and SNAP25. Yet, several exocytotic events, including apical exocytosis in epithelial cells, are insensitive to numerous clostridial NTs, suggesting the presence of SNARE-independent mechanisms of exocytosis. In this study we found that syntaxin 3, SNAP23, and a newly identified VAMP/brevin, tetanus neurotoxin (TeNT)-insensitive VAMP (TI-VAMP), are insensitive to clostridial NTs. In epithelial cells, TI-VAMP–containing vesicles were concentrated in the apical domain, and the protein was detected at the apical plasma membrane by immunogold labeling on ultrathin cryosections. Syntaxin 3 and SNAP23 were codistributed at the apical plasma membrane where they formed NEM-dependent SNARE complexes with TI-VAMP and cellubrevin. We suggest that TI-VAMP, SNAP23, and syntaxin 3 can participate in exocytotic processes at the apical plasma membrane of epithelial cells and, more generally, domain-specific exocytosis in clostridial NT-resistant pathways.

A Rab2 Mutant with Impaired GTPase Activity Stimulates Vesicle Formation from Pre-Golgi Intermediates

Rab2 immunolocalizes to pre-Golgi intermediates (vesicular-tubular clusters [VTCs]) that are the first site of segregation of anterograde- and retrograde-transported proteins and a major peripheral site for COPI recruitment. Our previous work showed that Rab2 Q65L (equivalent to Ras Q61L) inhibited endoplasmic reticulum (ER)-to-Golgi transport in vivo. In this study, the biochemical properties of Rab2 Q65L were analyzed. The mutant protein binds GDP and GTP and has a low GTP hydrolysis rate that suggests that Rab2 Q65L is predominantly in the GTP-bound–activated form. The purified protein arrests vesicular stomatitis virus glycoprotein transport from VTCs in an assay that reconstitutes ER-to-Golgi traffic. A quantitative binding assay was used to measure membrane binding of β-COP when incubated with the mutant. Unlike Rab2 that stimulates recruitment, Rab2 Q65L showed a dose-dependent decrease in membrane-associated β-COP when incubated with rapidly sedimenting membranes (ER, pre-Golgi, and Golgi). The mutant protein does not interfere with β-COP binding but stimulates the release of slowly sedimenting vesicles containing Rab2, β-COP, and p53/gp58 but lacking anterograde grade-directed cargo. To complement the biochemical results, we observed in a morphological assay that Rab2 Q65L caused vesiculation of VTCs that accumulated at 15°C. These data suggest that the Rab2 protein plays a role in the low-temperature–sensitive step that regulates membrane flow from VTCs to the Golgi complex and back to the ER.

The Phosphatidylinositol 3-Phosphate Binding Protein Vac1p Interacts with a Rab GTPase and a Sec1p Homologue to Facilitate Vesicle-mediated Vacuolar Protein Sorting

Activated GTP-bound Rab proteins are thought to interact with effectors to elicit vesicle targeting and fusion events. Vesicle-associated v-SNARE and target membrane t-SNARE proteins are also involved in vesicular transport. Little is known about the functional relationship between Rabs and SNARE protein complexes. We have constructed an activated allele of VPS21, a yeast Rab protein involved in vacuolar protein sorting, and demonstrated an allele-specific interaction between Vps21p and Vac1p. Vac1p was found to bind the Sec1p homologue Vps45p. Although no association between Vps21p and Vps45p was seen, a genetic interaction between VPS21 and VPS45 was observed. Vac1p contains a zinc-binding FYVE finger that may bind phosphatidylinositol 3-phosphate [PtdIns(3)P]. In other FYVE domain proteins, this motif and PtdIns(3)P are necessary for membrane association. Vac1 proteins with mutant FYVE fingers still associated with membranes but showed vacuolar protein sorting defects and reduced interactions with Vps45p and activated Vps21p. Vac1p membrane association was not dependent on PtdIns(3)P, Pep12p, Vps21p, Vps45p, or the PtdIns 3-kinase, Vps34p. Vac1p FYVE finger mutant missorting phenotypes were suppressed by a defective allele of VPS34. These data indicate that PtdIns(3)P may perform a regulatory role, possibly involved in mediating Vac1p protein–protein interactions. We propose that activated-Vps21p interacts with its effector, Vac1p, which interacts with Vps45p to regulate the Golgi to endosome SNARE complex.

Trafficking, Assembly, and Function of a Connexin43-Green Fluorescent Protein Chimera in Live Mammalian CellsV⃞

To examine the trafficking, assembly, and turnover of connexin43 (Cx43) in living cells, we used an enhanced red-shifted mutant of green fluorescent protein (GFP) to construct a Cx43-GFP chimera. When cDNA encoding Cx43-GFP was transfected into communication-competent normal rat kidney cells, Cx43-negative Madin–Darby canine kidney (MDCK) cells, or communication-deficient Neuro2A or HeLa cells, the fusion protein of predicted length was expressed, transported, and assembled into gap junctions that exhibited the classical pentalaminar profile. Dye transfer studies showed that Cx43-GFP formed functional gap junction channels when transfected into otherwise communication-deficient HeLa or Neuro2A cells. Live imaging of Cx43-GFP in MDCK cells revealed that many gap junction plaques remained relatively immobile, whereas others coalesced laterally within the plasma membrane. Time-lapse imaging of live MDCK cells also revealed that Cx43-GFP was transported via highly mobile transport intermediates that could be divided into two size classes of <0.5 μm and 0.5–1.5 μm. In some cases, the larger intracellular Cx43-GFP transport intermediates were observed to form from the internalization of gap junctions, whereas the smaller transport intermediates may represent other routes of trafficking to or from the plasma membrane. The localization of Cx43-GFP in two transport compartments suggests that the dynamic formation and turnover of connexins may involve at least two distinct pathways.

The Plant Vesicle-associated SNARE AtVTI1a Likely Mediates Vesicle Transport from the Trans-Golgi Network to the Prevacuolar Compartment

Membrane traffic in eukaryotic cells relies on recognition between v-SNAREs on transport vesicles and t-SNAREs on target membranes. Here we report the identification of AtVTI1a and AtVTI1b, two Arabidopsis homologues of the yeast v-SNARE Vti1p, which is required for multiple transport steps in yeast. AtVTI1a and AtVTI1b share 60% amino acid identity with one another and are 32 and 30% identical to the yeast protein, respectively. By suppressing defects found in specific strains of yeast vti1 temperature-sensitive mutants, we show that AtVTI1a can substitute for Vti1p in Golgi-to-prevacuolar compartment (PVC) transport, whereas AtVTI1b substitutes in two alternative pathways: the vacuolar import of alkaline phosphatase and the so-called cytosol-to-vacuole pathway used by aminopeptidase I. Both AtVTI1a and AtVTI1b are expressed in all major organs of Arabidopsis. Using subcellular fractionation and immunoelectron microscopy, we show that AtVTI1a colocalizes with the putative vacuolar cargo receptor AtELP on the trans-Golgi network and the PVC. AtVTI1a also colocalizes with the t-SNARE AtPEP12p to the PVC. In addition, AtVTI1a and AtPEP12p can be coimmunoprecipitated from plant cell extracts. We propose that AtVTI1a functions as a v-SNARE responsible for targeting AtELP-containing vesicles from the trans-Golgi network to the PVC, and that AtVTI1b is involved in a different membrane transport process.

Functional and Biochemical Analysis of the C2 Domains of Synaptotagmin IV

Synaptotagmins (Syts) are a family of vesicle proteins that have been implicated in both regulated neurosecretion and general membrane trafficking. Calcium-dependent interactions mediated through their C2 domains are proposed to contribute to the mechanism by which Syts trigger calcium-dependent neurotransmitter release. Syt IV is a novel member of the Syt family that is induced by cell depolarization and has a rapid rate of synthesis and a short half-life. Moreover, the C2A domain of Syt IV does not bind calcium. We have examined the biochemical and functional properties of the C2 domains of Syt IV. Consistent with its non–calcium binding properties, the C2A domain of Syt IV binds syntaxin isoforms in a calcium-independent manner. In neuroendocrine pheochromocytoma (PC12) cells, Syt IV colocalizes with Syt I in the tips of the neurites. Microinjection of the C2A domain reveals that calcium-independent interactions mediated through this domain of Syt IV inhibit calcium-mediated neurotransmitter release from PC12 cells. Conversely, the C2B domain of Syt IV contains calcium binding properties, which permit homo-oligomerization as well as hetero-oligomerization with Syt I. Our observation that different combinatorial interactions exist between Syt and syntaxin isoforms, coupled with the calcium stimulated hetero-oligomerization of Syt isoforms, suggests that the secretory machinery contains a vast repertoire of biochemical properties for sensing calcium and regulating neurotransmitter release accordingly.

Efficient Trafficking of TGN38 from the Endosome to the trans-Golgi Network Requires a Free Hydroxyl Group at Position 331 in the Cytosolic Domain

TGN38 is one of the few known resident integral membrane proteins of the trans-Golgi network (TGN). Since it cycles constitutively between the TGN and the plasma membrane, TGN38 is ideally suited as a model protein for the identification of post-Golgi trafficking motifs. Several studies, employing chimeric constructs to detect such motifs within the cytosolic domain of TGN38, have identified the sequence 333YQRL336 as an autonomous signal capable of localizing reporter proteins to the TGN. In addition, one group has found that an upstream serine residue, S331, may also play a role in TGN38 localization. However, the nature and degree of participation of S331 in the localization of TGN38 remain uncertain, and the effect has been studied in chimeric constructs only. Here we investigate the role of S331 in the context of full-length TGN38. Mutations that abolish the hydroxyl moiety at position 331 (A, D, and E) lead to missorting of endocytosed TGN38 to the lysosome. Conversely, mutation of S331 to T has little effect on the endocytic trafficking of TGN38. Together, these findings indicate that the S331 hydroxyl group has a direct or indirect effect on the ability of the cytosolic tail of TGN38 to interact with trafficking and/or sorting machinery at the level of the early endosome. In addition, mutation of S331 to either A or D results in increased levels of TGN38 at the cell surface. The results confirm that S331 plays a critical role in the intracellular trafficking of TGN38 and further reveal that TGN38 undergoes a signal-mediated trafficking step at the level of the endosome.

ATP- and Cytosol-dependent Release of Adaptor Proteins from Clathrin-coated Vesicles: A Dual Role for Hsc70

Clathrin-coated vesicles (CCV) mediate protein sorting and vesicular trafficking from the plasma membrane and the trans-Golgi network. Before delivery of the vesicle contents to the target organelles, the coat components, clathrin and adaptor protein complexes (APs), must be released. Previous work has established that hsc70/the uncoating ATPase mediates clathrin release in vitro without the release of APs. AP release has not been reconstituted in vitro, and nothing is known about the requirements for this reaction. We report a novel quantitative assay for the ATP- and cytosol- dependent release of APs from CCV. As expected, hsc70 is not sufficient for AP release; however, immunodepletion and reconstitution experiments establish that it is necessary. Interestingly, complete clathrin release is not a prerequisite for AP release, suggesting that hsc70 plays a dual role in recycling the constituents of the clathrin coat. This assay provides a functional basis for identification of the additional cytosolic factor(s) required for AP release.

Characterization of a Fourth Adaptor-related Protein Complex

Adaptor protein complexes (APs) function as vesicle coat components in different membrane traffic pathways; however, there are a number of pathways for which there is still no candidate coat. To find novel coat components related to AP complexes, we have searched the expressed sequence tag database and have identified, cloned, and sequenced a new member of each of the four AP subunit families. We have shown by a combination of coimmunoprecipitation and yeast two-hybrid analysis that these four proteins (ε, β4, μ4, and ς4) are components of a novel adaptor-like heterotetrameric complex, which we are calling AP-4. Immunofluorescence reveals that AP-4 is localized to ∼10–20 discrete dots in the perinuclear region of the cell. This pattern is disrupted by treating the cells with brefeldin A, indicating that, like other coat proteins, the association of AP-4 with membranes is regulated by the small GTPase ARF. Immunogold electron microscopy indicates that AP-4 is associated with nonclathrin-coated vesicles in the region of the trans-Golgi network. The μ4 subunit of the complex specifically interacts with a tyrosine-based sorting signal, indicating that, like the other three AP complexes, AP-4 is involved in the recognition and sorting of cargo proteins with tyrosine-based motifs. AP-4 is of relatively low abundance, but it is expressed ubiquitously, suggesting that it participates in a specialized trafficking pathway but one that is required in all cell types.

Fusion of Endosomes Involved in Synaptic Vesicle Recycling

Recycling of vesicles of the regulated secretory pathway presumably involves passage through an early endosomal compartment as an intermediate step. To learn more about the involvement of endosomes in the recycling of synaptic and secretory vesicles we studied in vitro fusion of early endosomes derived from pheochromocytoma (PC12) cells. Fusion was not affected by cleavage of the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins synaptobrevin and syntaxin 1 that operate at the exocytotic limb of the pathway. Furthermore, fusion was inhibited by the fast Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetra-acetic acid but not by the slow Ca2+ chelator EGTA. Endosome fusion was restored by the addition of Ca2+ with an optimum at a free Ca2+ concentration of 0.3 × 10−6 M. Other divalent cations did not substitute for Ca2+. A membrane-permeant EGTA derivative caused inhibition of fusion, which was reversed by addition of Ca2+. We conclude that the fusion of early endosomes participating in the recycling of synaptic and neurosecretory vesicles is mediated by a set of SNAREs distinct from those involved in exocytosis and requires the local release of Ca2+ from the endosomal interior.