SNAP-25 Palmitoylation and Plasma Membrane Targeting Require a Functional Secretory Pathway

Synaptosomal-associated protein of 25 kDa (SNAP-25) is a palmitoylated membrane protein essential for neurotransmitter release from synaptic terminals. We used neuronal cell lines to study the biosynthesis and posttranslational processing of SNAP-25 to investigate how palmitoylation contributes to the subcellular localization of the protein. SNAP-25 was synthesized as a soluble protein that underwent palmitoylation approximately 20 min after synthesis. Palmitoylation of the protein coincided with its stable membrane association. Treatment of cells with brefeldin A or other disrupters of transport inhibited palmitoylation of newly synthesized SNAP-25 and abolished membrane association. These results demonstrate that the processing of SNAP-25 and its targeting to the plasma membrane depend on an intact transport mechanism along the exocytic pathway. The kinetics of SNAP-25 palmitoylation and membrane association and the sensitivity of these parameters to brefeldin A suggest a novel trafficking pathway for targeting proteins to the plasma membrane. In vitro, SNAP-25 stably associated with membranes was not released from the membrane after chemical deacylation. We propose that palmitoylation of SNAP-25 is required for initial membrane targeting of the protein but that other interactions can maintain membrane association in the absence of fatty acylation.

Transduction of Basolateral-to-Apical Signals across Epithelial Cells: Ligand-stimulated Transcytosis of the Polymeric Immunoglobulin Receptor Requires Two Signals

Transcytosis of the polymeric immunoglobulin receptor (pIgR) is stimulated by binding of its ligand, dimeric IgA (dIgA). During this process, dIgA binding at the basolateral surface of the epithelial cell transmits a signal to the apical region of the cell, which in turn stimulates the transport of dIgA–pIgR complex from a postmicrotubule compartment to the apical surface. We have previously reported that the signal of stimulation was controlled by a protein-tyrosine kinase (PTK) activated upon dIgA binding. We now show that this signal of stimulation moves across the cell independently of pIgR movement or microtubules and acts through the tyrosine kinase activity by releasing Ca++ from inositol trisphosphate–sensitive intracellular stores. Surprisingly we have found that a second independent signal is required to achieve dIgA-stimulated transcytosis of pIgR. This second signal depends on dIgA binding to the pIgR solely at the basolateral surface and the ability of pIgR to dimerize. This enables pIgR molecules that have bound dIgA at the basolateral surface to respond to the signal of stimulation once they reach the postmicrotubule compartment. We propose that the use of two signals may be a general mechanism by which signaling receptors maintain specificity along their signaling and trafficking pathways.

ARF Is Required for Maintenance of Yeast Golgi and Endosome Structure and Function

ADP ribosylation factor (ARF) is thought to play a critical role in recruiting coatomer (COPI) to Golgi membranes to drive transport vesicle budding. Yeast strains harboring mutant COPI proteins exhibit defects in retrograde Golgi to endoplasmic reticulum protein transport and striking cargo-selective defects in anterograde endoplasmic reticulum to Golgi protein transport. To determine whether arf mutants exhibit similar phenotypes, the anterograde transport kinetics of multiple cargo proteins were examined in arf mutant cells, and, surprisingly, both COPI-dependent and COPI-independent cargo proteins exhibited comparable defects. Retrograde dilysine-mediated transport also appeared to be inefficient in the arf mutants, and coatomer mutants with no detectable anterograde transport defect exhibited a synthetic growth defect when combined with arf1Δ, supporting a role for ARF in retrograde transport. Remarkably, we found that early and medial Golgi glycosyltransferases localized to abnormally large ring-shaped structures. The endocytic marker FM4–64 also stained similar, but generally larger ring-shaped structures en route from the plasma membrane to the vacuole in arf mutants. Brefeldin A similarly perturbed endosome morphology and also inhibited transport of FM4–64 from endosomal structures to the vacuole. Electron microscopy of arf mutant cells revealed the presence of what appear to be hollow spheres of interconnected membrane tubules which likely correspond to the fluorescent ring structures. Together, these observations indicate that organelle morphology is significantly more affected than transport in the arf mutants, suggesting a fundamental role for ARF in regulating membrane dynamics. Possible mechanisms for producing this dramatic morphological change in intracellular organelles and its relation to the function of ARF in coat assembly are discussed.

Apiconuclear Organization of Microtubules Does Not Specify Protein Delivery from the Trans-Golgi Network to Different Membrane Domains in Polarized Epithelial Cells

In nonpolarized epithelial cells, microtubules originate from a broad perinuclear region coincident with the distribution of the Golgi complex and extend outward to the cell periphery (perinuclear [PN] organization). During development of epithelial cell polarity, microtubules reorganize to form long cortical filaments parallel to the lateral membrane, a meshwork of randomly oriented short filaments beneath the apical membrane, and short filaments at the base of the cell; the Golgi becomes localized above the nucleus in the subapical membrane cytoplasm (apiconuclear [AN] organization). The AN-type organization of microtubules is thought to be specialized in polarized epithelial cells to facilitate vesicle trafficking between the trans-Golgi Network (TGN) and the plasma membrane. We describe two clones of MDCK cells, which have different microtubule distributions: clone II/G cells, which gradually reorganize a PN-type distribution of microtubules and the Golgi complex to an AN-type during development of polarity, and clone II/J cells which maintain a PN-type organization. Both cell clones, however, exhibit identical steady-state polarity of apical and basolateral proteins. During development of cell surface polarity, both clones rapidly establish direct targeting pathways for newly synthesized gp80 and gp135/170, and E-cadherin between the TGN and apical and basolateral membrane, respectively; this occurs before development of the AN-type microtubule/Golgi organization in clone II/G cells. Exposure of both clone II/G and II/J cells to low temperature and nocodazole disrupts >99% of microtubules, resulting in: 1) 25–50% decrease in delivery of newly synthesized gp135/170 and E-cadherin to the apical and basolateral membrane, respectively, in both clone II/G and II/J cells, but with little or no missorting to the opposite membrane domain during all stages of polarity development; 2) ∼40% decrease in delivery of newly synthesized gp80 to the apical membrane with significant missorting to the basolateral membrane in newly established cultures of clone II/G and II/J cells; and 3) variable and nonspecific delivery of newly synthesized gp80 to both membrane domains in fully polarized cultures. These results define several classes of proteins that differ in their dependence on intact microtubules for efficient and specific targeting between the Golgi and plasma membrane domains.

Osmotically Induced Cell Volume Changes Alter Anterograde and Retrograde Transport, Golgi Structure, and COPI Dissociation

Physiological conditions that impinge on constitutive traffic and affect organelle structure are not known. We report that osmotically induced cell volume changes, which are known to occur under a variety of conditions, rapidly inhibited endoplasmic reticulum (ER)-to-Golgi transport in mammalian cells. Both ER export and ER Golgi intermediate compartment (ERGIC)-to-Golgi trafficking steps were blocked, but retrograde transport was active, and it mediated ERGIC and Golgi collapse into the ER. Extensive tubulation and relatively rapid Golgi resident redistribution were observed under hypo-osmotic conditions, whereas a slower redistribution of the same markers, without apparent tubulation, was observed under hyperosmotic conditions. The osmotic stress response correlated with the perturbation of COPI function, because both hypo- and hyperosmotic conditions slowed brefeldin A-induced dissociation of βCOP from Golgi membranes. Remarkably, Golgi residents reemerged after several hours of sustained incubation in hypotonic or hypertonic medium. Reemergence was independent of new protein synthesis but required PKC, an activity known to mediate cell volume recovery. Taken together these results indicate the existence of a coupling between cell volume and constitutive traffic that impacts organelle structure through independent effects on anterograde and retrograde flow and that involves, in part, modulation of COPI function.

Association of Myosin I Alpha with Endosomes and Lysosomes in Mammalian Cells

Myosin Is, which constitute a ubiquitous monomeric subclass of myosins with actin-based motor properties, are associated with plasma membrane and intracellular vesicles. Myosin Is have been proposed as key players for membrane trafficking in endocytosis or exocytosis. In the present paper we provide biochemical and immunoelectron microscopic evidence indicating that a pool of myosin I alpha (MMIα) is associated with endosomes and lysosomes. We show that the overproduction of MMIα or the production of nonfunctional truncated MMIα affects the distribution of the endocytic compartments. We also show that truncated brush border myosin I proteins, myosin Is that share 78% homology with MMIα, promote the dissociation of MMIα from vesicular membranes derived from endocytic compartments. The analysis at the ultrastructural level of cells producing these brush border myosin I truncated proteins shows that the delivery of the fluid phase markers from endosomes to lysosomes is impaired. MMIα might therefore be involved in membrane trafficking occurring between endosomes and lysosomes.

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.

The Formation of an Insulin-responsive Vesicular Cargo Compartment Is an Early Event in 3T3-L1 Adipocyte Differentiation

Differentiating 3T3-L1 cells exhibit a dramatic increase in the rate of insulin-stimulated glucose transport during their conversion from proliferating fibroblasts to nonproliferating adipocytes. On day 3 of 3T3-L1 cell differentiation, basal glucose transport and cell surface transferrin binding are markedly diminished. This occurs concomitant with the formation of a distinct insulin-responsive vesicular pool of intracellular glucose transporter 1 (GLUT1) and transferrin receptors as assessed by sucrose velocity gradients. The intracellular distribution of the insulin-responsive aminopeptidase is first readily detectable on day 3, and its gradient profile and response to insulin at this time are identical to that of GLUT1. With further time of differentiation, GLUT4 is expressed and targeted to the same insulin-responsive vesicles as the other three proteins. Our data are consistent with the notion that a distinct insulin-sensitive vesicular cargo compartment forms early during fat call differentiation and its formation precedes GLUT4 expression. The development of this compartment may result from the differentiation-dependent inhibition of constitutive GLUT1 and transferrin receptor trafficking such that there is a large increase in, or the new formation of, a population of postendosomal, insulin-responsive vesicles.

Membrane Tubule-mediated Reassembly and Maintenance of the Golgi Complex Is Disrupted by Phospholipase A2 Antagonists

Although membrane tubules can be found extending from, and associated with, the Golgi complex of eukaryotic cells, their physiological function has remained unclear. To gain insight into the biological significance of membrane tubules, we have developed methods for selectively preventing their formation. We show here that a broad range of phospholipase A2 (PLA2) antagonists not only arrest membrane tubule–mediated events that occur late in the assembly of the Golgi complex but also perturb its normal steady-state tubulovesicular architecture by inducing a reversible fragmentation into separate “mini-stacks.” In addition, we show that these same compounds prevent the formation of membrane tubules from Golgi stacks in an in vitro reconstitution system. This in vitro assay was further used to demonstrate that the relevant PLA2 activity originates from the cytoplasm. Taken together, these results demonstrate that Golgi membrane tubules, sensitive to potent and selective PLA2 antagonists, mediate both late events in the reassembly of the Golgi complex and the dynamic maintenance of its steady-state architecture. In addition, they implicate a role for cytoplasmic PLA2 enzymes in mediating these membrane trafficking events.

A Splice-Isoform of Vesicle-associated Membrane Protein-1 (VAMP-1) Contains a Mitochondrial Targeting Signal

Screening of a library derived from primary human endothelial cells revealed a novel human isoform of vesicle-associated membrane protein-1 (VAMP-1), a protein involved in the targeting and/or fusion of transport vesicles to their target membrane. We have termed this novel isoform VAMP-1B and designated the previously described isoform VAMP-1A. VAMP-1B appears to be an alternatively spliced form of VAMP-1. A similar rat splice variant of VAMP-1 (also termed VAMP-1B) has recently been reported. Five different cultured cell lines, from different lineages, all contained VAMP-1B but little or no detectable VAMP-1A mRNA, as assessed by PCR. In contrast, brain mRNA contained VAMP-1A but no VAMP-1B. The VAMP-1B sequence encodes a protein identical to VAMP-1A except for the carboxy-terminal five amino acids. VAMP-1 is anchored in the vesicle membrane by a carboxy-terminal hydrophobic sequence. In VAMP-1A the hydrophobic anchor is followed by a single threonine, which is the carboxy-terminal amino acid. In VAMP-1B the predicted hydrophobic membrane anchor is shortened by four amino acids, and the hydrophobic sequence is immediately followed by three charged amino acids, arginine-arginine-aspartic acid. Transfection of human endothelial cells with epitope-tagged VAMP-1B demonstrated that VAMP-1B was targeted to mitochondria whereas VAMP-1A was localized to the plasma membrane and endosome-like structures. Analysis of C-terminal mutations of VAMP-1B demonstrated that mitochondrial targeting depends both on the addition of positive charge at the C terminus and a shortened hydrophobic membrane anchor. These data suggest that mitochondria may be integrated, at least at a mechanistic level, to the vesicular trafficking pathways that govern protein movement between other organelles of the cell.

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.

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.

Ste6p Mutants Defective in Exit from the Endoplasmic Reticulum (ER) Reveal Aspects of an ER Quality Control Pathway in Saccharomyces cerevisiae

We are studying the intracellular trafficking of the multispanning membrane protein Ste6p, the a-factor transporter in Saccharomyces cerevisiae and a member of the ATP-binding cassette superfamily of proteins. In the present study, we have used Ste6p as model for studying the process of endoplasmic reticulum (ER) quality control, about which relatively little is known in yeast. We have identified three mutant forms of Ste6p that are aberrantly ER retained, as determined by immunofluorescence and subcellular fractionation. By pulse-chase metabolic labeling, we demonstrate that these mutants define two distinct classes. The single member of Class I, Ste6–166p, is highly unstable. We show that its degradation involves the ubiquitin–proteasome system, as indicated by its in vivo stabilization in certain ubiquitin–proteasome mutants or when cells are treated with the proteasome inhibitor drug MG132. The two Class II mutant proteins, Ste6–13p and Ste6–90p, are hyperstable relative to wild-type Ste6p and accumulate in the ER membrane. This represents the first report of a single protein in yeast for which distinct mutant forms can be channeled to different outcomes by the ER quality control system. We propose that these two classes of ER-retained Ste6p mutants may define distinct checkpoint steps in a linear pathway of ER quality control in yeast. In addition, a screen for high-copy suppressors of the mating defect of one of the ER-retained ste6 mutants has identified a proteasome subunit, Hrd2p/p97, previously implicated in the regulated degradation of wild-type hydroxymethylglutaryl-CoA reductase in the ER membrane.