Biomedical subjects

Martin Spiess

Publications and source records attributed to Martin Spiess.

15 recordsLinked to original sources

PubMed2006

The plug domain of yeast Sec61p is important for efficient protein translocation, but is not essential for cell viability.

The Sec61/SecY translocon mediates translocation of proteins across the membrane and integration of membrane proteins into the lipid bilayer. The structure of the translocon revealed a plug domain blocking the pore on the lumenal side. It was proposed to be important for gating the protein conducting channel and for maintaining the permeability barrier in its unoccupied state. Here, we analyzed in yeast the effect of introducing destabilizing point mutations in the plug domain or of its partial or complete deletion. Unexpectedly, even when the entire plug domain was deleted, cells were viable without growth phenotype. They showed an effect on signal sequence orientation of diagnostic signal-anchor proteins, a minor defect in cotranslational and a significant deficiency in posttranslational translocation. Steady-state levels of the mutant protein were reduced, and when coexpressed with wild-type Sec61p, the mutant lacking the plug competed poorly for complex partners. The results suggest that the plug is unlikely to be important for sealing the translocation pore in yeast but that it plays a role in stabilizing Sec61p during translocon formation.

Cell Survival↗

PubMed2005

Oligomerization and dissociation of AP-1 adaptors are regulated by cargo signals and by ArfGAP1-induced GTP hydrolysis.

The mechanism of AP-1/clathrin coat formation was analyzed using purified adaptor proteins and synthetic liposomes presenting tyrosine sorting signals. AP-1 adaptors recruited in the presence of Arf1.GTP and sorting signals were found to oligomerize to high-molecular-weight complexes even in the absence of clathrin. The appendage domains of the AP-1 adaptins were not required for oligomerization. On GTP hydrolysis induced by the GTPase-activating protein ArfGAP1, the complexes were disassembled and AP-1 dissociated from the membrane. AP-1 stimulated ArfGAP1 activity, suggesting a role of AP-1 in the regulation of the Arf1 "GTPase timer." In the presence of cytosol, AP-1 could be recruited to liposomes without sorting signals, consistent with the existence of docking factors in the cytosol. Under these conditions, however, AP-1 remained monomeric, and recruitment in the presence of GTP was short-lived. Sorting signals allowed stable recruitment and oligomerization also in the presence of cytosol. These results suggest a mechanism whereby initial assembly of AP-1 with Arf1.GTP and ArfGAP1 on the membrane stimulates Arf1 GTPase activity, whereas interaction with cargo induces oligomerization and reduces the rate of GTP hydrolysis, thus contributing to efficient cargo sorting.

Adaptor Protein Complex 2↗

PubMed2005

Proprotein convertase PC3 is not a transmembrane protein.

Proprotein convertase PC3 (also known as PC1) is an endopeptidase involved in proteolytic processing of peptide hormone precursors in granules of the regulated secretory pathway of endocrine cells. Lacking any extended hydrophobic segments, PC3 was considered to be a secretory protein only peripherally attached to the granule membrane. Recently, evidence has been presented that PC3 is a transmembrane protein with a 115-residue cytoplasmic domain and a membrane-spanning segment containing eight charged amino acids [Arnaoutova, I., et al. (2003) Biochemistry 42, 10445-10455]. Here, we analyzed the membrane topology of PC3 and of a PC3 construct containing a conventional transmembrane segment of 19 leucines. Alkaline extraction was performed to assess membrane integration. Exposure to the cytosol or to the ER lumen was tested by addition of C-terminal tags for phosphorylation or glycosylation, respectively. Protease sensitivity was assayed in permeabilized cells. The results show that the C-terminus of PC3 is translocated across the endoplasmic reticulum membrane. Furthermore, the proposed transmembrane segment of PC3 and a similar one of carboxypeptidase E did not stop polypeptide translocation when inserted into a stop-transfer tester construct. PC3 is thus not a transmembrane protein. These results have implications for the mechanism of granule sorting of PC3 as well as for the topology of PC2 and carboxypeptidase E, which have been reported to span the lipid membrane by homologous charged sequences.

Amino Acid Sequence↗

PubMed2005

Probing the environment of signal-anchor sequences during topogenesis in the endoplasmic reticulum.

Signal sequences for insertion of protein into the mammalian endoplasmic reticulum orient themselves in the translocon on the basis of their flanking charges. It has recently been shown that hydrophobic N-terminal signals initially insert head-on before they invert their orientation to translocate the C-terminus. The rate of inversion is reduced with the increasing hydrophobicity of the signal due to an increased affinity for the initial bound state at the translocon. To probe the environment of the signal while its orientation is determined, different hydrophobic residues were inserted at various positions throughout a uniform oligoleucine signal sequence and the constructs were expressed in transfected COS-7 cells. The resulting topologies revealed a strikingly symmetric position dependence specifically for bulky aromatic amino acids, reflecting the structure of a lipid bilayer. Maximal N-translocation was observed when the guest residues were placed at the N- or C-terminus of the hydrophobic sequence or in the very center, corresponding to the positions of highest expected affinity of the signal sequence as a membrane-spanning helix for the bilayer. The results support the model that during topogenesis in vivo the signal sequence is exposed to the lipid membrane.

Alanine↗

PubMed2005

Reconstitution of Rab4-dependent vesicle formation in vitro.

We have developed an in vitro assay to reconstitute the formation of endosomal recycling vesicles. To achieve specificity for endosomes as the donor organelle, cells are surface-biotinylated and allowed to endocytose for 10 min, after which the remaining surface-biotin is stripped off. The cells are then permeabilized and the cytosol washed away. Upon addition of exogenous cytosol and energy, sealed vesicles containing biotinylated recycling receptors are produced. Modification of the cytosol, for example, by immunodepletion or addition of purified proteins, allows the identification of proteins involved in vesicle formation. The results show that recycling is mediated by AP-1/clathrin-coated vesicles, requires Rab4, and is negatively regulated by rabaptin-5/rabex-5.

Animals↗

PubMed2004

Topogenesis of membrane proteins at the endoplasmic reticulum.

Most eukaryotic membrane proteins are cotranslationally integrated into the endoplasmic reticulum membrane by the Sec61 translocation complex. They are targeted to the translocon by hydrophobic signal sequences, which induce the translocation of either their N- or their C-terminal sequence. Signal sequence orientation is largely determined by charged residues flanking the apolar sequence (the positive-inside rule), folding properties of the N-terminal segment, and the hydrophobicity of the signal. Recent in vivo experiments suggest that N-terminal signals initially insert into the translocon head-on to yield a translocated N-terminus. Driven by a local electrical potential, the signal may invert its orientation and translocate the C-terminal sequence. Increased hydrophobicity slows down inversion by stabilizing the initial bound state. In vitro cross-linking studies indicate that signals rapidly contact lipids upon entering the translocon. Together with the recent crystal structure of the homologous SecYEbeta translocation complex of Methanococcus jannaschii, which did not reveal an obvious hydrophobic binding site for signals within the pore, a model emerges in which the translocon allows the lateral partitioning of hydrophobic segments between the aqueous pore and the lipid membrane. Signals may return into the pore for reorientation until translation is terminated. Subsequent transmembrane segments in multispanning proteins behave similarly and contribute to the overall topology of the protein.

Animals↗

PubMed2004

In vitro formation of recycling vesicles from endosomes requires adaptor protein-1/clathrin and is regulated by rab4 and the connector rabaptin-5.

The involvement of clathrin and associated adaptor proteins in receptor recycling from endosomes back to the plasma membrane is controversial. We have used an in vitro assay to identify the molecular requirements for the formation of recycling vesicles. Cells expressing the asialoglycoprotein receptor H1, a typical recycling receptor, were surface biotinylated and then allowed to endocytose for 10 min. After stripping away surface-biotin, the cells were permeabilized and the cytosol washed away. In a temperature-, cytosol-, and nucleotide-dependent manner, the formation of sealed vesicles containing biotinylated H1 could be reconstituted. Vesicle formation was strongly inhibited upon immunodepletion of adaptor protein (AP)-1, but not of AP-2 or AP-3, from the cytosol, and was restored by readdition of purified AP-1. Vesicle formation was stimulated by supplemented clathrin, but inhibited by brefeldin A, consistent with the involvement of ARF1 and a brefeldin-sensitive guanine nucleotide exchange factor. The GTPase rab4, but not rab5, was required to generate endosome-derived vesicles. Depletion of rabaptin-5/rabex-5, a known interactor of both rab4 and gamma-adaptin, stimulated and addition of the purified protein strongly inhibited vesicle production. The results indicate that recycling is mediated by AP-1/clathrin-coated vesicles and regulated by rab4 and rabaptin-5/rabex-5.

Adaptor Protein Complex 2↗

PubMed2004

Degradation of wild-type vasopressin precursor and pathogenic mutants by the proteasome.

Mutations in the gene encoding the antidiuretic hormone arginine vasopressin cause autosomal dominant neurogenic diabetes insipidus. Autoptic data in affected individuals suggest that the neurons expressing mutant vasopressin undergo selective degeneration. Expression studies have shown that the mutants are retained in the endoplasmic reticulum, but how this trafficking defect is linked to neurotoxicity is unknown. One possibility is that unsecreted mutant precursors, or degradation products thereof, are cytotoxic. We therefore investigated the fate of endoplasmic reticulum-retained pathogenic mutants. Our data show that the mutants are retrotranslocated to the cytosol and degraded by the proteasome. In the presence of proteasomal inhibitors, three distinct un- or deglycosylated cytosolic species of vasopressin precursors were stabilized: pre-pro-vasopressin, pro-vasopressin, and an N-terminally truncated form. In addition to the retrotranslocated forms, a fraction of the newly synthesized precursor was not translocated, but was synthesized into the cytosol due to inefficient function of the vasopressin signal peptide. As a result, cytosolic pre-pro-vasopressin and its degradation product were also recovered when wild-type vasopressin was expressed. Cytosolic forms of vasopressin might trigger cytotoxicity in vivo, as has been proposed in the case of prion protein, which also contains an inefficient N-terminal signal peptide.

Amino Acid Sequence↗

PubMed2004

Expression of regulated secretory proteins is sufficient to generate granule-like structures in constitutively secreting cells.

The formation of secretory granules and regulated secretion are generally assumed to occur only in specialized endocrine, neuronal, or exocrine cells. We discovered that regulated secretory proteins such as the hormone precursors pro-vasopressin, pro-oxytocin, and pro-opiomelanocortin, as well as the granins secretogranin II and chromogranin B but not the constitutive secretory protein alpha(1)-protease inhibitor, accumulate in granular structures at the Golgi and in the cell periphery in transfected COS-1 fibroblast cells. The accumulations were observed in 30-70% of the transfected cells expressing the pro-hormones and for virtually all of the cells expressing the granins. Similar structures were also generated in other cell lines believed to be lacking a regulated secretory pathway. The accumulations resembled secretory granules morphologically in immunofluorescence and electron microscopy. They were devoid of markers of the endoplasmic reticulum, endosomes, and lysosomes but in part stained positive for the trans-Golgi network marker TGN46, consistent with their formation at the trans-Golgi network. When different regulated proteins were coexpressed, they were frequently found in the same granules, whereas alpha(1)-protease inhibitor could not be detected in accumulations formed by secretogranin II, demonstrating segregation of regulated from constitutive secretory proteins. In pulse-chase experiments, significant intracellular storage of secretogranin II and chromogranin B was observed and secretion of retained secretogranin II was stimulated with the calcium ionophore A23187. The results suggest that expression of regulated cargo proteins is sufficient to generate structures that resemble secretory granules in the background of constitutively secreting cells, supporting earlier proposals on the mechanism of granule formation.

Animals↗

PubMed2003

Sec61p contributes to signal sequence orientation according to the positive-inside rule.

Protein targeting to the endoplasmic reticulum is mediated by signal or signal-anchor sequences. They also play an important role in protein topogenesis, because their orientation in the translocon determines whether their N- or C-terminal sequence is translocated. Signal orientation is primarily determined by charged residues flanking the hydrophobic core, whereby the more positive end is predominantly positioned to the cytoplasmic side of the membrane, a phenomenon known as the "positive-inside rule." We tested the role of conserved charged residues of Sec61p, the major component of the translocon in Saccharomyces cerevisiae, in orienting signals according to their flanking charges by site-directed mutagenesis by using diagnostic model proteins. Mutation of R67, R74, or E382 in Sec61p reduced C-terminal translocation of a signal-anchor protein with a positive N-terminal flanking sequence and increased it for signal-anchor proteins with positive C-terminal sequences. These mutations produced a stronger effect on substrates with greater charge difference across the hydrophobic core of the signal. For some of the substrates, a charge mutation in Sec61p had a similar effect as one in the substrate polypeptides. Although these three residues do not account for the entire charge effect in signal orientation, the results show that Sec61p contributes to the positive-inside rule.

Amino Acid Sequence↗

PubMed2003

Molecular mechanism of signal sequence orientation in the endoplasmic reticulum.

We have analyzed in vivo how model signal sequences are inserted and oriented in the membrane during cotranslational integration into the endoplasmic reticulum. The results are incompatible with the current models of retention of positive flanking charges or loop insertion of the polypeptide into the translocon. Instead they indicate that these N-terminal signals initially insert head-on with a cytoplasmic C-terminus before they invert their orientation to translocate the C-terminus. The rate of inversion increases with more positive N-terminal charge and is reduced with increasing hydrophobicity of the signal. Inversion may proceed for up to approximately 50 s, when it is terminated by a signal-independent process. These findings provide a mechanism for the topogenic effects of flanking charges as well as of signal hydrophobicity.

Animals↗

PubMed2002

Endoplasmic reticulum storage diseases.

The endoplasmic reticulum represents the cell's quality control site for accurate folding of secretory and membrane proteins. Quality control is achieved through the association of ER chaperones with unfolded or misfolded polypeptide chains. In the ER stress response, upregulation of chaperones occurs as a consequence of misfolded proteins accumulating in the ER lumen; if these proteins fail to assume their native structure, they are retained in the ER and targeted for degradation by the proteasome. ER storage diseases (ERSDs) are a group of genetically based disorders in which mutant proteins fail to pass the ER quality control. Because all eukaryotic cells contain the ER, the clinical phenotype of ERSDs is very heterogeneous. Disease may result from the mere lack of the mutant protein in question and/or may be caused indirectly by toxic effects of the misfolded protein or aggregates thereof on the cell. Additionally, the cell's reaction to the ER stress may include signaling pathways which are ultimately detrimental. Experimentally, ERSDs serve as models to study the cellular reactions to a variety of perturbations. In particular, understanding the links between ER stress and cell degeneration may give valuable insights into the pathogenesis of other diseases where the accumulation of indigestible toxic material leads to cell injury.

Endoplasmic Reticulum↗

PubMed2002

Ubiquitous 9-O-acetylation of sialoglycoproteins restricted to the Golgi complex.

9-O-Acetylation of sialic acid is known as a cell type-specific modification of secretory and plasma membrane glycoconjugates of higher vertebrates with important functions in modulating cell-cell recognition. Using a recombinant probe derived from influenza C virus hemagglutinin, we discovered 9-O-acetylated protein in the Golgi complex of various cell lines, most of which did not display 9-O-acetylated sialic acid on the cell surface. All cell lines expressed a sulfated glycoprotein of 50 kDa (sgp50) carrying 9-O-acetylated sialic acids, which was used as a model substrate. Like gp40, the major receptor for influenza C virus of Madin-Darby canine kidney I cells, sgp50 is 9-O-acetylated on O-linked glycans. However, gp40 was not 9-O-acetylated when expressed in Madin-Darby canine kidney II or COS-7 cells. The results demonstrate the existence of two 9-O-acetylation machineries for O-glycosylated proteins with distinct substrate specificities. The widespread occurrence of 9-O-acetylated protein in the Golgi furthermore suggests an additional intracellular role for this modification.

Acetylation↗

PubMed2002

Secretion of antithrombin is converted from nonpolarized to apical by exchanging its amino terminus for that of apically secreted family members.

The three members of the serpin family, corticosteroid binding globulin, alpha1-antitrypsin, and C1 inhibitor are secreted apically from Madin-Darby canine kidney (MDCK) cells, whereas two homologous family members, antithrombin and plasminogen activator inhibitor-1, are secreted in a nonpolarized fashion. cDNAs coding for chimeras composed of complementary portions of an apically targeted serpin and a nonsorted serpin were generated, expressed in MDCK cells, and the ratio between apical and basolateral secretion was analyzed. These experiments identified an amino-terminal sequence of corticosteroid binding globulin (residues 1-19) that is sufficient to direct a chimera with antithrombin mainly to the apical side. A deletion/mutagenesis analysis showed that no individual amino acid is absolutely required for the apical targeting ability of amino acids 1-30 of corticosteroid binding globulin. The corresponding amino-terminal sequences of alpha1-antitrypsin and C1 inhibitor were also sufficient to confer apical sorting. Based on our results we suggest that the apical targeting ability is encoded in the conformation of the protein.

Amino Acid Sequence↗

PubMed2002

ARF1.GTP, tyrosine-based signals, and phosphatidylinositol 4,5-bisphosphate constitute a minimal machinery to recruit the AP-1 clathrin adaptor to membranes.

At the trans-Golgi network, clathrin coats containing AP-1 adaptor complexes are formed in an ARF1-dependent manner, generating vesicles transporting cargo proteins to endosomes. The mechanism of site-specific targeting of AP-1 and the role of cargo are poorly understood. We have developed an in vitro assay to study the recruitment of purified AP-1 adaptors to chemically defined liposomes presenting peptides corresponding to tyrosine-based sorting motifs. AP-1 recruitment was found to be dependent on myristoylated ARF1, GTP or nonhydrolyzable GTP-analogs, tyrosine signals, and small amounts of phosphoinositides, most prominently phosphatidylinositol 4,5-bisphosphate, in the absence of any additional cytosolic or membrane bound proteins. AP-1 from cytosol could be recruited to a tyrosine signal independently of the lipid composition, but the rate of recruitment was increased by phosphatidylinositol 4,5-bisphosphate. The results thus indicate that cargo proteins are involved in coat recruitment and that the local lipid composition contributes to specifying the site of vesicle formation.

ADP-Ribosylation Factor 1↗