Promiscuity in Rab–SNARE Interactions

Fusion of post-Golgi secretory vesicles with the plasma membrane in yeast requires the function of a Rab protein, Sec4p, and a set of v- and t-SNAREs, the Snc, Sso, and Sec9 proteins. We have tested the hypothesis that a selective interaction between Sec4p and the exocytic SNAREs is responsible for ensuring that secretory vesicles fuse with the plasma membrane but not with intracellular organelles. Assembly of Sncp and Ssop into a SNARE complex is defective in a sec4-8 mutant strain. However, Snc2p binds in vivo to many other syntaxin-like t-SNAREs, and binding of Sncp to the endosomal/Golgi t-SNARE Tlg2p is also reduced in sec4-8 cells. In addition, binding of Sncp to Ssop is reduced by mutations in two other Rab genes and four non-Rab genes that block the secretory pathway before the formation of secretory vesicles. In an alternate approach to look for selective Rab–SNARE interactions, we report that the nucleotide-free form of Sec4p coimmunoprecipitates with Ssop. However, Rab–SNARE binding is nonselective, because the nucleotide-free forms of six Rab proteins bind with similar low efficiency to three SNARE proteins, Ssop, Pep12p, and Sncp. We conclude that Rabs and SNAREs do not cooperate to specify the target membrane.

Rapid regulated dense-core vesicle exocytosis requires the CAPS protein

Although many proteins essential for regulated neurotransmitter and peptide hormone secretion have been identified, little is understood about their precise roles at specific stages of the multistep pathway of exocytosis. To study the function of CAPS (Ca2+-dependent activator protein for secretion), a protein required for Ca2+-dependent exocytosis of dense-core vesicles, secretory responses in single rat melanotrophs were monitored by patch-clamp membrane capacitance measurements. Flash photolysis of caged Ca2+ elicited biphasic capacitance increases consisting of rapid and slow components with distinct Ca2+ dependencies. A threshold of ≈10 μM Ca2+ was required to trigger the slow component, while the rapid capacitance increase was recorded already at a intracellular Ca2+ activity < 10 μM. Both kinetic membrane capacitance components were abolished by botulinum neurotoxin B or E treatment, suggesting involvement of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor)-dependent vesicle fusion. The rapid but not the slow component was inhibited by CAPS antibody. These results were further clarified by immunocytochemical studies that revealed that CAPS was present on only a subset of dense-core vesicles. Overall, the results indicate that dense-core vesicle exocytosis in melanotrophs occurs by two parallel pathways. The faster pathway exhibits high sensitivity to Ca2+ and requires the presence of CAPS, which appears to act at a late stage in the secretory pathway.

Opposing mitogenic and anti-mitogenic actions of parathyroid hormone-related protein in vascular smooth muscle cells: A critical role for nuclear targeting

Parathyroid hormone-related protein (PTHrP) is a prohormone that is posttranslationally processed to a family of mature secretory forms, each of which has its own cognate receptor(s) on the cell surface that mediate the actions of PTHrP. In addition to being secreted via the classical secretory pathway and interacting with cell surface receptors in a paracrine/autocrine fashion, PTHrP appears to be able to enter the nucleus directly following translation and influence cellular events in an “intracrine” fashion. In this report, we demonstrate that PTHrP can be targeted to the nucleus in vascular smooth muscle cells, that this nuclear targeting is associated with a striking increase in mitogenesis, that this nuclear effect on proliferation is the diametric opposite of the effects of PTHrP resulting from interaction with cell surface receptors on vascular smooth muscle cells, and that the regions of the PTHrP sequence responsible for this nuclear targeting represent a classical bipartite nuclear localization signal. This report describes the activation of the cell cycle in association with nuclear localization of PTHrP in any cell type. These findings have important implications for the normal physiology of PTHrP in the many tissues which produce it, and suggest that gene delivery of PTHrP or modified variants may be useful in the management of atherosclerotic vascular disease.

Multifaceted Physiological Response Allows Yeast to Adapt to the Loss of the Signal Recognition Particle-dependent Protein-targeting PathwayD⃞

Translational control has recently been recognized as an important facet of adaptive responses to various stress conditions. We describe the adaptation response of the yeast Saccharomyces cerevisiae to the loss of one of two mechanisms to target proteins to the secretory pathway. Using inducible mutants that block the signal recognition particle (SRP) pathway, we find that cells demonstrate a physiological response to the loss of the SRP pathway that includes specific changes in global gene expression. Upon inducing the loss of the SRP pathway, SRP-dependent protein translocation is initially blocked, and cell growth is considerably slowed. Concomitantly, gene expression changes include the induction of heat shock genes and the repression of protein synthesis genes. Remarkably, within hours, the efficiency of protein sorting improves while cell growth remains slow in agreement with the persistent repression of protein synthesis genes. Our results suggest that heat shock gene induction serves to protect cells from mislocalized precursor proteins in the cytosol, whereas reduced protein synthesis helps to regain efficiency in protein sorting by reducing the load on the protein translocation apparatus. Thus, we suggest that cells trade speed in cell growth for fidelity in protein sorting to adjust to life without SRP.

Cellular mechanisms of β-amyloid production and secretion

The major constituent of senile plaques in Alzheimer’s disease is a 42-aa peptide, referred to as β-amyloid (Aβ). Aβ is generated from a family of differentially spliced, type-1 transmembrane domain (TM)-containing proteins, called APP, by endoproteolytic processing. The major, relatively ubiquitous pathway of APP metabolism in cell culture involves cleavage by α-secretase, which cleaves within the Aβ sequence, thus precluding Aβ formation and deposition. An alternate secretory pathway, enriched in neurons and brain, leads to cleavage of APP at the N terminus of the Aβ peptide by β-secretase, thus generating a cell-associated β-C-terminal fragment (β-CTF). A pathogenic mutation at codons 670/671 in APP (APP “Swedish”) leads to enhanced cleavage at the β-secretase scissile bond and increased Aβ formation. An inhibitor of vacuolar ATPases, bafilomycin, selectively inhibits the action of β-secretase in cell culture, suggesting a requirement for an acidic intracellular compartment for effective β-secretase cleavage of APP. β-CTF is cleaved in the TM domain by γ-secretase(s), generating both Aβ 1–40 (90%) and Aβ 1–42 (10%). Pathogenic mutations in APP at codon 717 (APP “London”) lead to an increased proportion of Aβ 1–42 being produced and secreted. Missense mutations in PS-1, localized to chromosome 14, are pathogenic in the majority of familial Alzheimer’s pedigrees. These mutations also lead to increased production of Aβ 1–42 over Aβ 1–40. Knockout of PS-1 in transgenic animals leads to significant inhibition of production of both Aβ 1–40 and Aβ 1–42 in primary cultures, indicating that PS-1 expression is important for γ-secretase cleavages. Peptide aldehyde inhibitors that block Aβ production by inhibiting γ-secretase cleavage of β-CTF have been discovered.

The metallochaperone Atox1 plays a critical role in perinatal copper homeostasis

Copper plays a fundamental role in the biochemistry of all aerobic organisms. The delivery of this metal to specific intracellular targets is mediated by metallochaperones. To elucidate the role of the metallochaperone Atox1, we analyzed mice with a disruption of the Atox1 locus. Atox1−/− mice failed to thrive immediately after birth, with 45% of pups dying before weaning. Surviving animals exhibited growth failure, skin laxity, hypopigmentation, and seizures because of perinatal copper deficiency. Maternal Atox1 deficiency markedly increased the severity of Atox1−/− phenotype, resulting in increased perinatal mortality as well as severe growth retardation and congenital malformations among surviving Atox1−/− progeny. Furthermore, Atox1-deficient cells accumulated high levels of intracellular copper, and metabolic studies indicated that this defect was because of impaired cellular copper efflux. Taken together, these data reveal a direct role for Atox1 in trafficking of intracellular copper to the secretory pathway of mammalian cells and demonstrate that this metallochaperone plays a critical role in perinatal copper homeostasis.

Fission Yeast Aip3p (spAip3p) Is Required for an Alternative Actin-directed Polarity Program

Aip3p is an actin-interacting protein that regulates cell polarity in budding yeast. The Schizosaccharomyces pombe-sequencing project recently led to the identification of a homologue of Aip3p that we have named spAip3p. Our results confirm that spAip3p is a true functional homologue of Aip3p. When expressed in budding yeast, spAip3p localizes similarly to Aip3p during the cell cycle and complements the cell polarity defects of an aip3Δ strain. Two-hybrid analysis shows that spAip3p interacts with actin similarly to Aip3p. In fission yeast, spAip3p localizes to both cell ends during interphase and later organizes into two rings at the site of cytokinesis. spAip3p localization to cell ends is dependent on microtubule cytoskeleton, its localization to the cell middle is dependent on actin cytoskeleton, and both patterns of localization require an operative secretory pathway. Overexpression of spAip3p disrupts the actin cytoskeleton and cell polarity, leading to morphologically aberrant cells. Fission yeast, which normally rely on the microtubule cytoskeleton to establish their polarity axis, can use the actin cytoskeleton in the absence of microtubule function to establish a new polarity axis, leading to the formation of branched cells. spAip3p localizes to, and is required for, branch formation, confirming its role in actin-directed polarized cell growth in both Schizosaccharomyces pombe and Saccharomyces cerevisiae.

Quantitative ER ↔ Golgi Transport Kinetics and Protein Separation upon Golgi Exit Revealed by Vesicular Integral Membrane Protein 36 Dynamics in Live CellsV⃞

To quantitatively investigate the trafficking of the transmembrane lectin VIP36 and its relation to cargo-containing transport carriers (TCs), we analyzed a C-terminal fluorescent-protein (FP) fusion, VIP36-SP-FP. When expressed at moderate levels, VIP36-SP-FP localized to the endoplasmic reticulum, Golgi apparatus, and intermediate transport structures, and colocalized with epitope-tagged VIP36. Temperature shift and pharmacological experiments indicated VIP36-SP-FP recycled in the early secretory pathway, exhibiting trafficking representative of a class of transmembrane cargo receptors, including the closely related lectin ERGIC53. VIP36-SP-FP trafficking structures comprised tubules and globular elements, which translocated in a saltatory manner. Simultaneous visualization of anterograde secretory cargo and VIP36-SP-FP indicated that the globular structures were pre-Golgi carriers, and that VIP36-SP-FP segregated from cargo within the Golgi and was not included in post-Golgi TCs. Organelle-specific bleach experiments directly measured the exchange of VIP36-SP-FP between the Golgi and endoplasmic reticulum (ER). Fitting a two-compartment model to the recovery data predicted first order rate constants of 1.22 ± 0.44%/min for ER → Golgi, and 7.68 ± 1.94%/min for Golgi → ER transport, revealing a half-time of 113 ± 70 min for leaving the ER and 1.67 ± 0.45 min for leaving the Golgi, and accounting for the measured steady-state distribution of VIP36-SP-FP (13% Golgi/87% ER). Perturbing transport with AlF4− treatment altered VIP36-SP-GFP distribution and changed the rate constants. The parameters of the model suggest that relatively small differences in the first order rate constants, perhaps manifested in subtle differences in the tendency to enter distinct TCs, result in large differences in the steady-state localization of secretory components.

A Novel Quality Control Compartment Derived from the Endoplasmic Reticulum

Degradation of proteins that, because of improper or suboptimal processing, are retained in the endoplasmic reticulum (ER) involves retrotranslocation to reach the cytosolic ubiquitin-proteasome machinery. We found that substrates of this pathway, the precursor of human asialoglycoprotein receptor H2a and free heavy chains of murine class I major histocompatibility complex (MHC), accumulate in a novel preGolgi compartment that is adjacent to but not overlapping with the centrosome, the Golgi complex, and the ER-to-Golgi intermediate compartment (ERGIC). On its way to degradation, H2a associated increasingly after synthesis with the ER translocon Sec61. Nevertheless, it remained in the secretory pathway upon proteasomal inhibition, suggesting that its retrotranslocation must be tightly coupled to the degradation process. In the presence of proteasomal inhibitors, the ER chaperones calreticulin and calnexin, but not BiP, PDI, or glycoprotein glucosyltransferase, concentrate in the subcellular region of the novel compartment. The “quality control” compartment is possibly a subcompartment of the ER. It depends on microtubules but is insensitive to brefeldin A. We discuss the possibility that it is also the site for concentration and retrotranslocation of proteins that, like the mutant cystic fibrosis transmembrane conductance regulator, are transported to the cytosol, where they form large aggregates, the “aggresomes.”

Split invertase polypeptides form functional complexes in the yeast periplasm in vivo.

The assembly of functional proteins from fragments in vivo has been recently described for several proteins, including the secreted maltose binding protein in Escherichia coli. Here we demonstrate for the first time that split gene products can function within the eukaryotic secretory system. Saccharomyces cerevisiae strains able to use sucrose produce the enzyme invertase, which is targeted by a signal peptide to the central secretory pathway and the periplasmic space. Using this enzyme as a model we find the following: (i) Polypeptide fragments of invertase, each containing a signal peptide, are independently translocated into the endoplasmic reticulum (ER) are modified by glycosylation, and travel the entire secretory pathway reaching the yeast periplasm. (ii) Simultaneous expression of independently translated and translocated overlapping fragments of invertase leads to the formation of an enzymatically active complex, whereas individually expressed fragments exhibit no activity. (iii) An active invertase complex is assembled in the ER, is targeted to the yeast periplasm, and is biologically functional, as judged by its ability to facilitate growth on sucrose as a single carbon source. These observation are discussed in relation to protein folding and assembly in the ER and to the trafficking of proteins through the secretory pathway.

Insulin-secreting non-islet cells are resistant to autoimmune destruction.

Transgenic nonobese diabetic mice were created in which insulin expression was targeted to proopiomelanocortin-expressing pituitary cells. Proopiomelanocortin-expressing intermediate lobe pituitary cells efficiently secrete fully processed, mature insulin via a regulated secretory pathway, similar to islet beta cells. However, in contrast to the insulin-producing islet beta cells, the insulin-producing intermediate lobe pituitaries are not targeted or destroyed by cells of the immune system. Transplantation of the transgenic intermediate lobe tissues into diabetic nonobese diabetic mice resulted in the restoration of near-normoglycemia and the reversal of diabetic symptoms. The absence of autoimmunity in intermediate lobe pituitary cells engineered to secrete bona fide insulin raises the potential of these cell types for beta-cell replacement therapy for the treatment of insulin-dependent diabetes mellitus.

Isolation of the human PC6 gene encoding the putative host protease for HIV-1 gp160 processing in CD4+ T lymphocytes.

Production of infectious HIV-1 virions is dependent on the processing of envelope glycoprotein gp160 by a host cell protease. The protease in human CD4+ T lymphocytes has not been unequivocally identified, yet members of the family of mammalian subtilisin-like protein convertases (SPCs), which are soluble or membrane-bound proteases of the secretory pathway, best fulfill the criteria. These proteases are required for proprotein maturation and cleave at paired basic amino acid motifs in numerous cellular and viral glycoprotein precursors, both in vivo and in vitro. To identify the gp160 processing protease, we have used reverse transcription-PCR and Northern blot analyses to ascertain the spectrum of SPC proteases in human CD4+ T cells. We have cloned novel members of the SPC family, known as the human PC6 genes. Two isoforms of the hPC6 protease are expressed in human T cells, hPC6A and the larger hPC6B. The patterns of SPC gene expression in human T cells has been compared with the furin-defective LoVo cell line, both of which are competent in the production of infectious HIV virions. This comparison led to the conclusion that the hPC6 gene products are the most likely candidates for the host cell protease responsible for HIV-1 gp160 processing in human CD4+ T cells.

A mutant cytochrome b5 with a lengthened membrane anchor escapes from the endoplasmic reticulum and reaches the plasma membrane.

Many resident membrane proteins of the endoplasmic reticulum (ER) do not have known retrieval sequences. Among these are the so-called tail-anchored proteins, which are bound to membranes by a hydrophobic tail close to the C terminus and have most of their sequence as a cytosolically exposed N-terminal domain. Because ER tail-anchored proteins generally have short (< or = 17 residues) hydrophobic domains, we tested whether this feature is important for localization, using cytochrome b5 as a model. The hydrophobic domain of cytochrome b5 was lengthened by insertion of five amino acids (ILAAV), and the localization of the mutant was analyzed by immunofluorescence in transiently transfected mammalian cells. While the wild-type cytochrome was localized to the ER, the mutant was relocated to the surface. This relocation was not due to the specific sequence introduced, as demonstrated by the ER localization of a second mutant, in which the original length of the membrane anchor was restored, while maintaining the inserted ILAAV sequence. Experiments with brefeldin A and with cycloheximide demonstrated that the extended anchor mutant reached the plasma membrane by transport along the secretory pathway. We conclude that the short membrane anchor of cytochrome b5 is important for its ER residency, and we discuss the relevance of this finding for other ER tail-anchored proteins.

rSec6 and rSec8, mammalian homologs of yeast proteins essential for secretion.

Many of the molecules necessary for neurotransmission are homologous to proteins involved in the Golgi-to-plasma membrane stage of the yeast secretory pathway. Of 15 genes known to be essential for the later stages of vesicle trafficking in yeast, 7 have no identified mammalian homologs. These include the yeast SEC6, SEC8, and SEC15 genes, whose products are constituents of a 19.5S particle that interacts with the GTP-binding protein Sec4p. Here we report the sequences of rSec6 and rSec8, rat homologs of Sec6p and Sec8p. The rSec6 cDNA is predicted to encode an 87-kDa protein with 22% amino acid identity to Sec6p, and the rSec8 cDNA is predicted to encode a 110-kDa protein which is 20% identical to Sec8p. Northern blot analysis indicates that rSec6 and rSec8 are expressed in similar tissues. Immunodetection reveals that rSec8 is part of a soluble 17S particle in brain. COS cell cotransfection studies demonstrate that rSec8 colocalizes with the GTP-binding protein Rab3a and syntaxin 1a, two proteins involved in synaptic vesicle docking and fusion at the presynaptic terminal. These data suggest that rSec8 is a component of a high molecular weight complex which may participate in the regulation of vesicle docking and fusion in brain.

Enteropathogenic Escherichia coli contains a putative type III secretion system necessary for the export of proteins involved in attaching and effacing lesion formation.

Enteropathogenic Escherichia coli (EPEC) causes a characteristic histopathology in intestinal epithelial cells called the attaching and effacing lesion. Although the histopathological lesion is well described the bacterial factors responsible for it are poorly characterized. We have identified four EPEC chromosomal genes whose predicted protein sequences are similar to components of a recently described secretory pathway (type III) responsible for exporting proteins lacking a typical signal sequence. We have designated the genes sepA, sepB, sepC, and sepD (sep, for secretion of E. coli proteins). The predicted Sep polypeptides are similar to the Lcr (low calcium response) and Ysc (yersinia secretion) proteins of Yersinia species and the Mxi (membrane expression of invasion plasmid antigens) and Spa (surface presentation of antigens) regions of Shigella flexneri. Culture supernatants of EPEC strain E2348/69 contain several polypeptides ranging in size from 110 kDa to 19 kDa. Proteins of comparable size were recognized by human convalescent serum from a volunteer experimentally infected with strain E2348/69. A sepB mutant of EPEC secreted only the 110-kDa polypeptide and was defective in the formation of attaching and effacing lesions and protein-tyrosine phosphorylation in tissue culture cells. These phenotypes were restored upon complementation with a plasmid carrying an intact sepB gene. These data suggest that the EPEC Sep proteins are components of a type III secretory apparatus necessary for the export of virulence determinants.