Late Secretory Pathway Research Articles

Role of PACS-1 in trafficking of human cytomegalovirus glycoprotein B and virus production.

The final envelopment of herpesviruses during assembly of new virions is thought to occur by the budding of core viral particles into a late secretory pathway organelle, the trans-Golgi network (TGN), or an associated endosomal compartment. Several herpesvirus envelope glycoproteins have been previously shown to localize to the TGN when expressed independently from other viral proteins. In at least some cases this TGN localization has been shown to be dependent on clusters of acidic residues within their cytoplasmic domains. Similar acidic cluster motifs are found in endogenous membrane proteins that also localize to the TGN. These acidic cluster motifs interact with PACS-1, a connector protein that is required for the trafficking of proteins containing such motifs from endosomes to the TGN. We show here that PACS-1 interacts with the cytoplasmic domain of the HCMV envelope glycoprotein B (gB) and that PACS-1 function is required for normal TGN localization of HCMV gB. Furthermore, inhibition of PACS-1 activity in infected cells leads to a decrease in HCMV titer, whereas an increase in expression of functional PACS-1 leads to an increase in HCMV titer, suggesting that PACS-1 is required for efficient production of HCMV.

Requirement for neo1p in retrograde transport from the Golgi complex to the endoplasmic reticulum.

Neo1p from Saccharomyces cerevisiae is an essential P-type ATPase and potential aminophospholipid translocase (flippase) in the Drs2p family. We have previously implicated Drs2p in protein transport steps in the late secretory pathway requiring ADP-ribosylation factor (ARF) and clathrin. Here, we present evidence that epitope-tagged Neo1p localizes to the endoplasmic reticulum (ER) and Golgi complex and is required for a retrograde transport pathway between these organelles. Using conditional alleles of NEO1, we find that loss of Neo1p function causes cargo-specific defects in anterograde protein transport early in the secretory pathway and perturbs glycosylation in the Golgi complex. Rer1-GFP, a protein that cycles between the ER and Golgi complex in COPI and COPII vesicles, is mislocalized to the vacuole in neo1-ts at the nonpermissive temperature. These phenotypes suggest that the anterograde protein transport defect is a secondary consequence of a defect in a COPI-dependent retrograde pathway. We propose that loss of lipid asymmetry in the cis Golgi perturbs retrograde protein transport to the ER.

An Autocatalytic Cleavage in the C Terminus of the Human MUC2 Mucin Occurs at the Low pH of the Late Secretory Pathway

During purification of a recombinant MUC2 C terminus expressed in CHO-K1 cells, the protein was partly cleaved when buffers with a pH of 6.0 were used. When buffers with higher pH values were used, less cleavage was found. Disulfide bonds held the two fragments generated together as these were only observed after reduction. Edman sequencing of the C-terminal 110-kDa fragment revealed that the cleavage had occurred at an Asp-Pro bond, a site described previously to generate the so-called "link peptide" after disulfide bond reduction. In vitro studies on the conditions for cleavage showed that it occurred in a time-dependent manner at a pH below 6.0. Furthermore, the reaction was not enzyme-mediated as it occurred in pure preparations of the MUC2 C terminus and was not inhibited by protease inhibitors. When expressed in the mucin producing cell line LS 174T, the C terminus was cleaved to a higher extent compared with the CHO-K1 cells. Neutralizing the secretory pathway with either NH(4)Cl or bafilomycin A1 inhibited this cleavage. Altogether, our results suggest that the cleavage is an autocatalytic reaction that occurs in the acidic environment of the late secretory pathway. Furthermore, the cleavage produced a new, reactive C terminus that has the potential to attach the mucin to itself or other molecules. Because a pH below 6 can be reached in the late secretory pathway and on mucosal surfaces, the cleavage and possible cross-linking are likely to be of biological importance.

Lipid rafts and assembly of enveloped viruses.

The plasma membrane, late secretory pathway and endosomal compartments contain detergent-insoluble raft microdomains that are enriched in sphingolipids and cholesterol. Rafts are currently an intensively studied topic of cell biology, and raft involvement has been implicated in numerous cellular processes. A number of recent reports have localized structural proteins of several enveloped viruses to rafts, thus raising the possibility that rafts also play a role in the assembly and budding of viruses, but what exactly that role might be is still unknown.

Brefeldin A-induced prosomatostatin N-glycosylation in AtT20 cells

Prosomatostatin, the precursor of the hormone somatostatin, harbors an N-glycosylation site in its prodomain that has never been shown to be modified by the N-oligosaccharyl transferase (OST) of the endoplasmic reticulum (ER). The addition of Brefeldin A (BFA) to prosomatostatin transfected AtT20 cells leads to a quantitative glycosylation of the prohormone. Upon removal of the BFA the glycosylated hormone precursor is not deglycosylated, and is secreted after maturation of its oligosaccharide chain in the late secretory pathway. In addition, a significant proportion of the glycosylated hormone precursor remains in the cell. Since BFA is known to induce an effective collapse of the Golgi complex into the ER, the hypothesis that a prolonged exposure to the ER glycosylation machinery is responsible for the glycosylation was tested. No N-glycosylation was detected using a coupled in vitro transcription–translation system in the presence of canine pancreatic microsomes, indicating that rapid transit through the ER does not explain the lack of glycosylation observed in vivo in the absence of BFA. These observations show that co-translational glycosylation by OST becomes possible due to a still unidentified modification in the luminal environment brought about by the coalescence of the Golgi into the ER caused by BFA.

Hormonal control of cerebral amyloidogenesis in Alzheimer's diseases

Estrogen reduces the risk of Alzheimer disease (AD) in postmenopausal women, β‐amyloid (Aβ) burden in animal models of AD, and secretion of Aβ from neuronal cultures. The biological basis for these effects remains unknown. Aβ is proteolytically derived from the β‐amyloid precursor protein (βAPP) within the secretory pathway by distinct enzymatic activities known as β‐ and gamma‐secretase. Aggregated Aβ peptides are found predominantly within extraneuronal space and are believed to initiate toxic and inflammatory cascades leading to neuronal death. The major population of secreted Aβ peptides is generated within the trans‐Golgi‐network (TGN), also the major site of βAPP residence in neurons at steady state. Utilizing cell‐free systems derived from both neuroblastoma cells and primary neurons, we demonstrate that 17β‐estradiol (17β‐E2) stimulates formation of vesicles containing βAPP, from the TGN. Accelerated βAPP trafficking precludes maximal Aβ generation within the TGN. 17β‐E2 appears to modulate TGN phospholipid levels, particularly those of phosphatidylinositol, and recruit soluble trafficking factors, such as Rab11, to the TGN. Together, these results suggest that estrogen may exert its anti‐Aβ effects by regulating βAPP trafficking within the late secretory pathway. These results suggest a novel mechanism through which 17β‐E2 may act in estrogen‐responsive tissues and illustrate how altering the kinetics of a protein's transport can influence its metabolic fate.

Estrogen Lowers Alzheimer β-Amyloid Generation by Stimulating trans-Golgi Network Vesicle Biogenesis

Estrogen reduces the risk of Alzheimer's disease in post-menopausal women, beta-amyloid (Abeta) burden in animal models of Alzheimer's disease, and secretion of Abeta from neuronal cultures. The biological basis for these effects remains unknown. Here, utilizing cell-free systems derived from both neuroblastoma cells and primary neurons, we demonstrate that 17beta-estradiol (17beta-E2) stimulates formation of vesicles containing the beta-amyloid precursor protein (betaAPP) from the trans-Golgi network (TGN). Accelerated betaAPP trafficking precludes maximal Abeta generation within the TGN. 17beta-E2 appears to modulate TGN phospholipid levels, particularly those of phosphatidylinositol, and to recruit soluble trafficking factors, such as Rab11, to the TGN. Together, these results suggest that estrogen may exert its anti-Abeta effects by regulating betaAPP trafficking within the late secretory pathway. These results suggest a novel mechanism through which 17beta-E2 may act in estrogen-responsive tissues and illustrate how altering the kinetics of the transport of a protein can influence its metabolic fate.

Furin initiates gelsolin familial amyloidosis in the Golgi through a defect in Ca(2+) stabilization.

Hereditary familial amyloidosis of Finnish type (FAF) leading to amyloid in the peripheral and central nervous systems stems from deposition of a 71 residue fragment generated from the D187N/Y variants of plasma gelsolin by two sequential endoproteolytic events. We identify the protease accomplishing the first cleavage as furin, a proprotein convertase. Endoproteolysis of plasma gelsolin occurs in the trans-Golgi network due to the inability of the FAF variants to bind and be stabilized by Ca(2+). Secretion and processing of the FAF variants by furin can be uncoupled by blocking the convergence of the exocytic pathway transporting plasma gelsolin and the endocytic recycling of furin. We propose that coincidence of membrane trafficking pathways contributes to the development of proteolysis-initiated amyloid disease.

Ubiquitin is required for sorting to the vacuole of the yeast general amino acid permease, Gap1.

In yeast, ubiquitin plays a central role in proteolysis of a multitude of proteins and serves also as a signal for endocytosis of many plasma membrane proteins. We showed previously that ubiquitination of the general amino acid permease (Gap1) is essential to its endocytosis followed by vacuolar degradation. These processes occur when NH(4)(+), a preferential source of nitrogen, is added to cells growing on proline or urea, i.e. less favored nitrogen sources. In this study, we show that Gap1 is ubiquitinated on two lysine residues in the cytosolic N terminus (positions 9 and 16). A mutant Gap1 in which both lysines are mutated (Gap1(K9K16)) remains fully stable at the plasma membrane after NH(4)(+) addition. Furthermore, each of the two lysines harbors a poly-ubiquitin chain in which ubiquitin is linked to the lysine 63 of the preceding ubiquitin. The Gap1(K9) and Gap1(K16) mutants, in which a single lysine is mutated, are down-regulated in response to NH(4)(+) although more slowly. In proline-grown cells lacking Npr1, a protein kinase involved in the control of Gap1 trafficking, newly synthesized Gap1 is sorted from the Golgi to the vacuole without passing through the plasma membrane (accompanying article, De Craene, J.-O., Soetens, O., and André, B. (2001) J. Biol. Chem. 276, 43939-43948). We show here that ubiquitination of Gap1 is also required for this direct sorting to the vacuole. In an npr1Delta mutant, neosynthesized Gap1(K9K16) is rerouted to and accumulates at the plasma membrane. Finally, Bul1 and Bul2, two proteins interacting with Npi1/Rsp5, are essential to ubiquitination and down-regulation of cell-surface Gap1, as well as to sorting of neosynthesized Gap1 to the vacuole, as occurs in an npr1Delta mutant. Our results reveal a novel role of ubiquitin in the control of Gap1 trafficking, i.e. direct sorting from the late secretory pathway to the vacuole. This result reinforces the growing evidence that ubiquitin plays an important role not only in internalization of plasma membrane proteins but also in their sorting in the endosomes and/or trans-Golgi.

Clathrin-binding proteins: Got a motif? Join the network!

Clathrin plays a key function in membrane and protein trafficking through the endocytic and late secretory pathways. Its role as a molecular scaffold that drives formation of transport vesicles requires binding to a number of proteins with distinct functional and structural properties. Recent studies have revealed that most of these proteins interact with clathrin through surprisingly simple, linear arrangements of acidic and hydrophobic amino acid residues. This article discusses the different types of clathrin-binding proteins and motifs as well as the physiological significance of these proteins in clathrin-dependent events.

Evidence for a satellite secretory pathway in neuronal dendritic spines.

Long-term information storage within the brain requires the synthesis of new proteins and their use in synapse-specific modifications [1]. Recently, we demonstrated that translation sites for the local synthesis of integral membrane and secretory proteins occur within distal dendritic spines [2]. It remains unresolved, however, whether a complete secretory pathway, including Golgi and trans Golgi network-like membranes, exists near synapses for the local transport and processing of newly synthesized proteins. Here, we report evidence of a satellite secretory pathway in distal dendritic spines and distal dendrites of the mammalian brain. Membranes analogous to early (RER and ERGIC), middle (Golgi cisternae), and late (TGN) secretory pathway compartments are present within dendritic spines and in distal dendrites. Local synthesis, processing, and transport of newly translated integral membrane and secretory proteins may thus provide the molecular basis for synapse-specific modifications during long-term information storage in the brain.

Yeast secretory expression of insulin precursors

Since the 1980s, recombinant human insulin for the treatment of diabetes mellitus has been produced using either the yeast Saccharomyces cerevisiae or the prokaryote Escherichia coli. Here, development of the insulin secretory expression system in S. cerevisiae and its subsequent optimisation is described. Expression of proinsulin in S. cerevisiae does not result in efficient secretion of proinsulin or insulin. However, expression of a cDNA encoding a proinsulin-like molecule with deletion of threonine(B30) as a fusion protein with the S. cerevisiae alpha-factor prepro-peptide (leader), followed either by replacement of the human proinsulin C-peptide with a small C-peptide (e.g. AAK), or by direct fusion of lysine(B29) to glycine(A1), results in the efficient secretion of folded single-chain proinsulin-like molecules to the culture supernatant. The secreted single-chain insulin precursor can then be purified and subsequently converted to human insulin by tryptic transpeptidation in organic aqueous medium in the presence of a threonine ester. The leader confers secretory competence to the insulin precursor, and constructed (synthetic) leaders have been developed for efficient secretory expression of the insulin precursor in the yeasts S. cerevisiae and Pichia pastories. The Kex2 endoprotease, specific for dibasic sites, cleaves the leader-insulin precursor fusion protein in the late secretory pathway and the folded insulin precursor is secreted to the culture supernatant. However, the Kex2 endoprotease processing of the pro-peptide-insulin precursor fusion protein is incomplete and a significant part of the pro-peptide-insulin precursor fusion protein is secreted to the culture supernatant in a hyperglycosylated form. A spacer peptide localised between the leader and the insulin precursor has been developed to optimise Kex2 endoprotease processing and insulin precursor fermentation yield.

Yeast VSM1 encodes a v-SNARE binding protein that may act as a negative regulator of constitutive exocytosis.

We have screened for proteins that interact with v-SNAREs of the late secretory pathway in the yeast Saccharomyces cerevisiae. A novel protein, designated Vsm1, binds tightly to the Snc2 v-SNARE in the two-hybrid system and can be coimmunoprecipitated with Snc1 or Snc2 from solubilized yeast cell extracts. Disruption of the VSM1 gene results in an increase of proteins secreted into the medium but does not affect the processing or secretion of invertase. In contrast, VSM1 overexpression in cells which bear a temperature-sensitive mutation in the Sec9 t-SNARE (sec9-4 cells) results in the accumulation of non-invertase-containing low-density secretory vesicles, inhibits cell growth and the secretion of proteins into the medium, and blocks rescue of the temperature-sensitive phenotype by SNC1 overexpression. Yet, VSM1 overexpression does not affect yeast bearing a sec9-7 allele which, in contrast to sec9-4, encodes a t-SNARE protein capable of forming a stable SNARE complex in vitro at restrictive temperatures. On the basis of these results, we propose that Vsm1 is a novel v-SNARE-interacting protein that appears to act as negative regulator of constitutive exocytosis. Moreover, this regulation appears specific to one of two parallel exocytic paths which are operant in yeast cells.

Molecular bases for the recognition of tyrosine-based sorting signals.

Targeting of transmembrane proteins to different compartments of the endocytic and late (post-Golgi) secretory pathways is largely dependent upon sorting signals contained within the cytosolic domains of the proteins (reviewed in reference [19][1], [24][2]). The signals are thought to interact with

AP-4, a Novel Protein Complex Related to Clathrin Adaptors

Here we report the identification and characterization of AP-4, a novel protein complex related to the heterotetrameric AP-1, AP-2, and AP-3 adaptors that mediate protein sorting in the endocytic and late secretory pathways. The key to the identification of this complex was the cloning and sequencing of two widely expressed, mammalian cDNAs encoding new homologs of the adaptor beta and sigma subunits named beta4 and sigma4, respectively. An antibody to beta4 recognized in human cells an approximately 83-kDa polypeptide that exists in both soluble and membrane-associated forms. Gel filtration, sedimentation velocity, and immunoprecipitation experiments revealed that beta4 is a component of a multisubunit complex (AP-4) that also contains the sigma4 polypeptide and two additional adaptor subunit homologs named mu4 (mu-ARP2) and epsilon. Immunofluorescence analyses showed that AP-4 is associated with the trans-Golgi network or an adjacent structure and that this association is sensitive to the drug brefeldin A. We propose that, like the related AP-1, AP-2, and AP-3 complexes, AP-4 plays a role in signal-mediated trafficking of integral membrane proteins in mammalian cells.

Chs6p-dependent anterograde transport of Chs3p from the chitosome to the plasma membrane in Saccharomyces cerevisiae.

Chitin synthase III (CSIII), an enzyme required to form a chitin ring in the nascent division septum of Saccharomyces cerevisiae, may be transported to the cell surface in a regulated manner. Chs3p, the catalytic subunit of CSIII, requires the product of CHS6 to be transported to or activated at the cell surface. We find that chs6Delta strains have morphological abnormalities similar to those of chs3 mutants. Subcellular fractionation and indirect immunofluorescence indicate that Chs3p distribution is altered in chs6 mutant cells. Order-of-function experiments using end4-1 (endocytosis-defective) and chs6 mutants indicate that Chs6p is required for anterograde transport of Chs3p from an internal endosome-like membrane compartment, the chitosome, to the plasma membrane. As a result, chs6 strains accumulate Chs3p in chitosomes. Chs1p, a distinct chitin synthase that acts during or after cell separation, is transported normally in chs6 mutants, suggesting that Chs1p and Chs3p are independently packaged during protein transport through the late secretory pathway.

Smy1p, a kinesin-related protein that does not require microtubules.

We have previously reported that a defect in Myo2p, a myosin in budding yeast (Saccharomyces cerevisiae), can be partially corrected by overexpression of Smy1p, which is by sequence a kinesin-related protein (Lillie, S.H., and S.S. Brown. 1992. Nature. 356:358- 361). Such a functional link between putative actin- and microtubule-based motors is surprising, so here we have tested the prediction that Smy1p indeed acts as a microtubule-based motor. Unexpectedly, we found that abolition of microtubules by nocodazole does not interfere with the ability of Smy1p to correct the mutant Myo2p defect, nor does it interfere with the ability of Smy1p to localize properly. In addition, other perturbations of microtubules, such as treatment with benomyl or introduction of tubulin mutations, do not exacerbate the Myo2p defect. Furthermore, a mutation in SMY1 strongly predicted to destroy motor activity does not destroy Smy1p function. We have also observed a genetic interaction between SMY1 and two of the late SEC mutations, sec2 and sec4. This indicates that Smy1p can play a role even when Myo2p is wild type, and that Smy1p acts at a specific step of the late secretory pathway. We conclude that Smy1p does not act as a microtubule-based motor to localize properly or to compensate for defective Myo2p, but that it must instead act in some novel way.

Physiological regulation of membrane protein sorting late in the secretory pathway of Saccharomyces cerevisiae.

In mammalian cells, extracellular signals can regulate the delivery of particular proteins to the plasma membrane. We have discovered a novel example of regulated protein sorting in the late secretory pathway of Saccharomyces cerevisiae. In yeast cells grown on either ammonia or urea medium, the general amino acid permease (Gap1p) is transported from the Golgi complex to the plasma membrane, whereas, in cells grown on glutamate medium, Gap1p is transported from the Golgi to the vacuole. We have also found that sorting of Gap1p in the Golgi is controlled by SEC13, a gene previously shown to encode a component of the COPII vesicle coat. In sec13 mutants grown on ammonia, Gap1p is transported from the Golgi to the vacuole, instead of to the plasma membrane. Deletion of PEP12, a gene required for vesicular transport from the Golgi to the prevacuolar compartment, counteracts the effect of the sec13 mutation and partially restores Gap1p transport to the plasma membrane. Together, these studies demonstrate that both a nitrogen-sensing mechanism and Sec13p control Gap1p transport from the Golgi to the plasma membrane.

A Novel RING Finger Protein, Vps8p, Functionally Interacts with the Small GTPase, Vps21p, to Facilitate Soluble Vacuolar Protein Localization

Genetic analyses of vacuolar protein sorting in Saccharomyces cerevisiae have uncovered a large number of mutants (vps) that missort and secrete soluble vacuolar hydrolases. Here we report the characterization of the gene product affected in one of these mutants, Vps8p. Polyclonal antiserum raised against a trpE-Vps8 fusion protein specifically detects a 134-kDa protein in labeled yeast cell extracts. Subcellular fractionation studies demonstrate that Vps8p is distributed between a low speed membrane pellet fraction and a high speed membrane pellet fraction. The lack of a hydrophobic domain in Vps8p suggests that Vps8p peripherally associates with a membrane(s). This association was found to depend on the function of Vps21p, a member of the Rab/Ypt/Sec4 family of small GTPases. In vps21 null mutant cells, Vps8p is found in the cytosol. In addition, overexpression of Vps21p partially suppresses a vps8 null mutant, indicating that Vps8p and Vps21p functionally interact. Vps8p contains a C-terminal cysteine-rich region that conforms to the H2 variant of the RING finger Zn2+ binding motif. Truncation of this C-terminal region partially compromises Vps8p function. While vps8 null mutant strains missort and secrete soluble vacuolar hydrolases, the integral vacuolar membrane protein, alkaline phosphatase (ALP), is sorted to the vacuole and matured normally. In addition, when vps8 mutants are combined with endocytic or late secretory pathway mutants (end3 or sec1, respectively), ALP is still delivered to the vacuole. These observations indicate that ALP is sorted to the vacuole in a Vps8p-independent manner, possibly via an alternative vesicle carrier.

Differential trafficking and timed localization of two chitin synthase proteins, Chs2p and Chs3p.

The deposition of the polysaccharide chitin in the Saccharomyces cerevisiae cell wall is temporally and spatially regulated. Chitin synthase III (Chs3p) synthesizes a ring of chitin at the onset of bud emergence, marking the base of the incipient bud. At the end of mitosis, chitin synthase II (Chs2p) deposits a disk of chitin in the mother-bud neck, forming the primary division septum. Using indirect immunofluorescence microscopy, we have found that these two integral membrane proteins localize to the mother-bud neck at distinct times during the cell cycle. Chs2p is found at the neck at the end of mitosis, whereas Chs3p localizes to a ring on the surface of cells about to undergo bud emergence and in the mother-bud neck of small-budded cells. Cell synchronization and pulse-chase experiments suggest that the timing of Chs2p localization results from cell cycle-specific synthesis coupled to rapid degradation. Chs2p degradation depends on the vacuolar protease encoded by PEP4, indicating that Chs2p is destroyed in the vacuole. Temperature-sensitive mutations that block either the late secretory pathway (sec1-1) or the internalization step of endocytosis (end4-1) also prevent Chs2p degradation. In contrast, Chs3p is synthesized constitutively and is metabolically stable, indicating that Chs2p and Chs3p are subject to different modes of regulation. Differential centrifugation experiments show that a significant proportion of Chs3p resides in an internal compartment that may correspond to a vesicular species called the chitosome (Leal-Morales, C.A., C.E. Bracker, and S. Bartnicki-Garcia. 1988, Proc. Natl. Acad. Sci. USA. 85:8516-8520; Flores Martinez, A., and J. Schwencke. 1988. Biochim. Biophys. Acta. 946:328-336). Fractionation of membranes prepared from mutants defective in internalization (end3-1 and end4-1) indicate that the Chs3p-containing vesicles are endocytically derived. Collectively, these data suggest that the trafficking of Chs2p and Chs3p diverges after endocytosis; Chs3p is not delivered to the vacuole, but instead may be recycled.