Secretion ofSaccharomyces cerevisiaeKiller Toxin: Processing of the Glycosylated Precursor

Killer toxin secretion was blocked at the restrictive temperature inSaccharomyces cerevisiae secmutants with conditional defects in theS. cerevisiaesecretory pathway leading to accumulation of endoplasmic reticulum (sec18), Golgi (sec7), or secretory vesicles (sec1). A 43,000-molecular-weight (43K) glycosylated protoxin was found by pulse-labeling in allsecmutants at the restrictive temperature. Insec18the protoxin was stable after a chase; but insec7andsec1the protoxin was unstable, and insec111K toxin was detected in cell lysates. The chymotrypsin inhibitor tosyl-l-phenylalanyl chloromethyl ketone (TPCK) blocked toxin secretion in vivo in wild-type cells by inhibiting protoxin cleavage. The unstable protoxin in wild-type and insec7andsec1cells at the restrictive temperature was stabilized by TPCK, suggesting that the protoxin cleavage was post-sec18and was mediated by a TPCK-inhibitable protease. Protoxin glycosylation was inhibited by tunicamycin, and a 36K protoxin was detected in inhibited cells. This 36K protoxin was processed, but toxin secretion was reduced 10-fold. We examined twokexmutants defective in toxin secretion; both synthesized a 43K protoxin, which was stable inkex1but unstable inkex2. Protoxin stability inkex1 kex2double mutants indicated the orderkex1→kex2in the protoxin processing pathway. TPCK did not block protoxin instability inkex2mutants. This suggested that theKEX1- andKEX2-dependent steps preceded thesec7Golgi block. We attempted to localize the protoxin inS. cerevisiaecells. Use of an in vitro rabbit reticulocyte-dog pancreas microsomal membrane system indicated that protoxin synthesized in vitro could be inserted into and glycosylated by the microsomal membranes. This membrane-associated protoxin was protected from trypsin proteolysis. Pulse-chased cells or spheroplasts, with or without TPCK, failed to secrete protoxin. The protoxin may not be secreted into the lumen of the endoplasmic reticulum, but may remain membrane associated and may require endoproteolytic cleavage for toxin secretion.

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Secretion of Saccharomyces cerevisiae Killer Toxin: Processing of the Glycosylated Precursor

Molecular and Cellular Biology ◽

10.1128/mcb.3.8.1362-1370.1983 ◽

1983 ◽

Vol 3 (8) ◽

pp. 1362-1370

Author(s):

H. Bussey ◽

D. Saville ◽

D. Greene ◽

D. J. Tipper ◽

K. A. Bostian

Keyword(s):

Saccharomyces Cerevisiae ◽

Endoplasmic Reticulum ◽

Secretory Pathway ◽

Killer Toxin ◽

Restrictive Temperature ◽

Wild Type ◽

Endoproteolytic Cleavage ◽

Toxin Secretion

Killer toxin secretion was blocked at the restrictive temperature in Saccharomyces cerevisiae sec mutants with conditional defects in the S. cerevisiae secretory pathway leading to accumulation of endoplasmic reticulum ( sec18 ), Golgi ( sec7 ), or secretory vesicles ( sec1 ). A 43,000-molecular-weight (43K) glycosylated protoxin was found by pulse-labeling in all sec mutants at the restrictive temperature. In sec18 the protoxin was stable after a chase; but in sec7 and sec1 the protoxin was unstable, and in sec1 11K toxin was detected in cell lysates. The chymotrypsin inhibitor tosyl- l -phenylalanyl chloromethyl ketone (TPCK) blocked toxin secretion in vivo in wild-type cells by inhibiting protoxin cleavage. The unstable protoxin in wild-type and in sec7 and sec1 cells at the restrictive temperature was stabilized by TPCK, suggesting that the protoxin cleavage was post- sec18 and was mediated by a TPCK-inhibitable protease. Protoxin glycosylation was inhibited by tunicamycin, and a 36K protoxin was detected in inhibited cells. This 36K protoxin was processed, but toxin secretion was reduced 10-fold. We examined two kex mutants defective in toxin secretion; both synthesized a 43K protoxin, which was stable in kex1 but unstable in kex2 . Protoxin stability in kex1 kex2 double mutants indicated the order kex1 → kex2 in the protoxin processing pathway. TPCK did not block protoxin instability in kex2 mutants. This suggested that the KEX1 - and KEX2 -dependent steps preceded the sec7 Golgi block. We attempted to localize the protoxin in S. cerevisiae cells. Use of an in vitro rabbit reticulocyte-dog pancreas microsomal membrane system indicated that protoxin synthesized in vitro could be inserted into and glycosylated by the microsomal membranes. This membrane-associated protoxin was protected from trypsin proteolysis. Pulse-chased cells or spheroplasts, with or without TPCK, failed to secrete protoxin. The protoxin may not be secreted into the lumen of the endoplasmic reticulum, but may remain membrane associated and may require endoproteolytic cleavage for toxin secretion.

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Reconstitution of protein transport from the endoplasmic reticulum to the Golgi complex in yeast: the acceptor Golgi compartment is defective in the sec23 mutant.

The Journal of Cell Biology ◽

10.1083/jcb.107.4.1465 ◽

1988 ◽

Vol 107 (4) ◽

pp. 1465-1476 ◽

Cited By ~ 102

Author(s):

H Ruohola ◽

A K Kabcenell ◽

S Ferro-Novick

Keyword(s):

Endoplasmic Reticulum ◽

Secretory Pathway ◽

Protein Transport ◽

Yeast Cells ◽

Restrictive Temperature ◽

Transport Reaction ◽

Permeabilized Cells ◽

Alpha Factor

Using either permeabilized cells or microsomes we have reconstituted the early events of the yeast secretory pathway in vitro. In the first stage of the reaction approximately 50-70% of the prepro-alpha-factor, synthesized in a yeast translation lysate, is translocated into the endoplasmic reticulum (ER) of permeabilized yeast cells or directly into yeast microsomes. In the second stage of the reaction 48-66% of the ER form of alpha-factor (26,000 D) is then converted to the high molecular weight Golgi form in the presence of ATP, soluble factors and an acceptor membrane fraction; GTP gamma S inhibits this transport reaction. Donor, acceptor, and soluble fractions can be separated in this assay. This has enabled us to determine the defective fraction in sec23, a secretory mutant that blocks ER to Golgi transport in vivo. When fractions were prepared from mutant cells grown at the permissive or restrictive temperature and then assayed in vitro, the acceptor Golgi fraction was found to be defective.

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The In Vivo Association of BiP with Newly Synthesized Proteins Is Dependent on the Rate and Stability of Folding and Not Simply on the Presence of Sequences That Can Bind to BiP

The Journal of Cell Biology ◽

10.1083/jcb.144.1.21 ◽

1999 ◽

Vol 144 (1) ◽

pp. 21-30 ◽

Cited By ~ 56

Author(s):

Rachel Hellman ◽

Marc Vanhove ◽

Annabelle Lejeune ◽

Fred J. Stevens ◽

Linda M. Hendershot

Keyword(s):

Secretory Pathway ◽

Variable Region ◽

Wild Type ◽

Mutant Proteins ◽

Protein Subunits ◽

Hsp70 Family ◽

Nascent Chain ◽

Kinetic Traps

Immunoglobulin heavy chain-binding protein (BiP) is a member of the hsp70 family of chaperones and one of the most abundant proteins in the ER lumen. It is known to interact transiently with many nascent proteins as they enter the ER and more stably with protein subunits produced in stoichiometric excess or with mutant proteins. However, there also exists a large number of secretory pathway proteins that do not apparently interact with BiP. To begin to understand what controls the likelihood that a nascent protein entering the ER will associate with BiP, we have examined the in vivo folding of a murine λI immunoglobulin (Ig) light chain (LC). This LC is composed of two Ig domains that can fold independent of the other and that each possess multiple potential BiP-binding sequences. To detect BiP binding to the LC during folding, we used BiP ATPase mutants, which bind irreversibly to proteins, as “kinetic traps.” Although both the wild-type and mutant BiP clearly associated with the unoxidized variable region domain, we were unable to detect binding of either BiP protein to the constant region domain. A combination of in vivo and in vitro folding studies revealed that the constant domain folds rapidly and stably even in the absence of an intradomain disulfide bond. Thus, the simple presence of a BiP-binding site on a nascent chain does not ensure that BiP will bind and play a role in its folding. Instead, it appears that the rate and stability of protein folding determines whether or not a particular site is recognized, with BiP preferentially binding to proteins that fold slowly or somewhat unstably.

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Compare HBsAg retention in the endoplasmic reticulum and the ER stress between the cells transfected or infected by the wild type and pre-S HBV mutants using in vitro transfection and in vivo infection of FRG mice

Journal of Hepatology ◽

10.1016/s0168-8278(18)31799-9 ◽

2018 ◽

Vol 68 ◽

pp. S767

Author(s):

J.Y.-J. Liang ◽

J.-C. Wu ◽

Y. Chi ◽

C.-P. Sun ◽

C.-W. Su ◽

...

Keyword(s):

Endoplasmic Reticulum ◽

Er Stress ◽

Wild Type ◽

In Vitro Transfection

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Overexpression of wild-type and mutant ARF1 and ARF6: distinct perturbations of nonoverlapping membrane compartments.

The Journal of Cell Biology ◽

10.1083/jcb.128.6.1003 ◽

1995 ◽

Vol 128 (6) ◽

pp. 1003-1017 ◽

Cited By ~ 259

Author(s):

P J Peters ◽

V W Hsu ◽

C E Ooi ◽

D Finazzi ◽

S B Teal ◽

...

Keyword(s):

Plasma Membrane ◽

Golgi Complex ◽

Secretory Pathway ◽

Membrane System ◽

Endocytic Pathway ◽

Coat Proteins ◽

Wild Type ◽

Gtp Binding ◽

Defective Mutant

The ARF GTP binding proteins are believed to function as regulators of membrane traffic in the secretory pathway. While the ARF1 protein has been shown in vitro to mediate the membrane interaction of the cytosolic coat proteins coatomer (COP1) and gamma-adaptin with the Golgi complex, the functions of the other ARF proteins have not been defined. Here, we show by transient transfection with epitope-tagged ARFs, that whereas ARF1 is localized to the Golgi complex and can be shown to affect predictably the assembly of COP1 and gamma-adaptin with Golgi membranes in cells, ARF6 is localized to the endosomal/plasma membrane system and has no effect on these Golgi-associated coat proteins. By immuno-electron microscopy, the wild-type ARF6 protein is observed along the plasma membrane and associated with endosomes, and overexpression of ARF6 does not appear to alter the morphology of the peripheral membrane system. In contrast, overexpression of ARF6 mutants predicted either to hydrolyze or bind GTP poorly shifts the distribution of ARF6 and affects the structure of the endocytic pathway. The GTP hydrolysis-defective mutant is localized to the plasma membrane and its overexpression results in a profound induction of extensive plasma membrane vaginations and a depletion of endosomes. Conversely, the GTP binding-defective ARF6 mutant is present exclusively in endosomal structures, and its overexpression results in a massive accumulation of coated endocytic structures.

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PRP18, a protein required for the second reaction in pre-mRNA splicing.

Molecular and Cellular Biology ◽

10.1128/mcb.10.1.324 ◽

1990 ◽

Vol 10 (1) ◽

pp. 324-332 ◽

Cited By ~ 36

Author(s):

U Vijayraghavan ◽

J Abelson

Keyword(s):

Micrococcal Nuclease ◽

Heat Inactivation ◽

Temperature Sensitive ◽

Restrictive Temperature ◽

Wild Type ◽

Exon 1 ◽

In Vitro Splicing

We have investigated the role of a novel temperature-sensitive splicing mutation, prp18. We had previously demonstrated that an accumulation of the lariat intermediate of splicing occurred at the restrictive temperature in vivo. We have now used the yeast in vitro splicing system to show that extracts from this mutant strain are heat labile for the second reaction of splicing. The heat inactivation of prp18 extracts results from loss of activity of an exchangeable component. Inactivated prp18 extracts are complemented by heat-inactivated extracts from other mutants or by fractions from wild-type extracts. In heat-inactivated prp18 extracts, 40S splicing complexes containing lariat intermediate and exon 1 can assemble. The intermediates in this 40S complex can be chased to products by complementing extracts in the presence of ATP. Both complementation of extracts and chasing of the isolated prp18 spliceosomes takes place with micrococcal nuclease-treated extracts. Furthermore, the complementation profile with fractions of wild-type extracts indicates that the splicing defect results from a mutation in a previously designated factor required for the second step of splicing. The isolation of this mutant as temperature-sensitive lethal has also facilitated cloning of the wild-type allele by complementation.

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SEC62 encodes a putative membrane protein required for protein translocation into the yeast endoplasmic reticulum.

The Journal of Cell Biology ◽

10.1083/jcb.109.6.2653 ◽

1989 ◽

Vol 109 (6) ◽

pp. 2653-2664 ◽

Cited By ~ 115

Author(s):

R J Deshaies ◽

R Schekman

Keyword(s):

Endoplasmic Reticulum ◽

Protein Translocation ◽

Signal Sequence ◽

Alpha Helix ◽

Wild Type ◽

Cytosol Fraction ◽

Arginine Residues ◽

Mutant Cells

Yeast sec62 mutant cells are defective in the translocation of several secretory precursor proteins into the lumen of the endoplasmic reticulum (Rothblatt et al., 1989). The deficiency, which is most restrictive for alpha-factor precursor (pp alpha F) and preprocarboxypeptidase Y, has been reproduced in vitro. Membranes isolated from mutant cells display low and labile translocation activity with pp alpha F translated in a wild-type cytosol fraction. The defect is unique to the membrane fraction because cytosol from mutant cells supports translocation into membranes from wild-type yeast. Invertase assembly is only partly affected by the sec62 mutation in vivo and is nearly normal with mutant membranes in vitro. A potential membrane location for the SEC62 gene product is supported by evaluation of the molecular clone. DNA sequence analysis reveals a 32-kD protein with no obvious NH2-terminal signal sequence but with two domains of sufficient length and hydrophobicity to span a lipid bilayer. Sec62p is predicted to display significant NH2- and COOH-terminal hydrophilic domains on the cytoplasmic surface of the ER membrane. The last 30 amino acids of the COOH terminus may form an alpha-helix with 14 lysine and arginine residues arranged uniformly about the helix. This domain may allow Sec62p to interact with other proteins of the putative translocation complex.

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Mss4 does not function as an exchange factor for Rab in endoplasmic reticulum to Golgi transport.

Molecular Biology of the Cell ◽

10.1091/mbc.8.7.1305 ◽

1997 ◽

Vol 8 (7) ◽

pp. 1305-1316 ◽

Cited By ~ 42

Author(s):

C Nuoffer ◽

S K Wu ◽

C Dascher ◽

W E Balch

Keyword(s):

Endoplasmic Reticulum ◽

Secretory Pathway ◽

Rab Proteins ◽

Guanine Nucleotide ◽

Nucleotide Exchange ◽

Rab Protein ◽

Guanine Nucleotide Exchange ◽

Golgi Transport

Mss4 and its yeast homologue, Dss4, have been proposed to function as guanine nucleotide exchange factors (GEFs) for a subset of Rab proteins in the secretory pathway. We have previously shown that Rab1A mutants defective in GTP-binding potently inhibit endoplasmic reticulum to Golgi transport, presumably by sequestering an unknown GEF regulating its function. We now demonstrate that these mutants stably associate with Mss4 both in vivo and in vitro and that Mss4 effectively neutralizes the inhibitory activity of the Rab1A mutants. An equivalent Rab3A mutant (Rab3A[N135I]), a Rab protein specifically involved in regulated secretion at the cell surface, associates with Mss4 as efficiently as the Rab1A[N124I] mutant. Although Rab3A[N135I] prevents the ability of Mss4 to neutralize the inhibitory effects of Rab1A mutants on transport, it has no effect on Rab1 function or endoplasmic reticulum to Golgi transport. Furthermore, quantitative immunodepletion of Mss4 fails to inhibit transport in vitro. We conclude that Mss4 and its yeast homologue, Dss4, are not GEFs mediating activation of Rab, but rather, they interact with the transient guanine nucleotide-free state, defining a new class of Ras-superfamily GTPase effectors that function as guanine nucleotide-free chaperones (GFCs).

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In vivo and in vitro analyses of amygdalar function reveal a role for copper

Journal of Neurophysiology ◽

10.1152/jn.00631.2013 ◽

2014 ◽

Vol 111 (10) ◽

pp. 1927-1939 ◽

Cited By ~ 26

Author(s):

E. D. Gaier ◽

R. M. Rodriguiz ◽

J. Zhou ◽

M. Ralle ◽

W. C. Wetsel ◽

...

Keyword(s):

Fear Conditioning ◽

Secretory Pathway ◽

Pyramidal Neurons ◽

Physiological Role ◽

Long Term Potentiation ◽

Single Copy ◽

Inhibitory Effect ◽

Wild Type

Mice with a single copy of the peptide amidating monooxygenase ( Pam) gene (PAM+/−) are impaired in contextual and cued fear conditioning. These abnormalities coincide with deficient long-term potentiation (LTP) at excitatory thalamic afferent synapses onto pyramidal neurons in the lateral amygdala. Slice recordings from PAM+/− mice identified an increase in GABAergic tone (Gaier ED, Rodriguiz RM, Ma XM, Sivaramakrishnan S, Bousquet-Moore D, Wetsel WC, Eipper BA, Mains RE. J Neurosci 30: 13656–13669, 2010). Biochemical data indicate a tissue-specific deficit in Cu content in the amygdala; amygdalar expression of Atox-1 and Atp7a, essential for transport of Cu into the secretory pathway, is reduced in PAM+/− mice. When PAM+/− mice were fed a diet supplemented with Cu, the impairments in fear conditioning were reversed, and LTP was normalized in amygdala slice recordings. A role for endogenous Cu in amygdalar LTP was established by the inhibitory effect of a brief incubation of wild-type slices with bathocuproine disulfonate, a highly selective, cell-impermeant Cu chelator. Interestingly, bath-applied CuSO4 had no effect on excitatory currents but reversibly potentiated the disynaptic inhibitory current. Bath-applied CuSO4 was sufficient to potentiate wild-type amygdala afferent synapses. The ability of dietary Cu to affect signaling in pathways that govern fear-based behaviors supports an essential physiological role for Cu in amygdalar function at both the synaptic and behavioral levels. This work is relevant to neurological and psychiatric disorders in which disturbed Cu homeostasis could contribute to altered synaptic transmission, including Wilson's, Menkes, Alzheimer's, and prion-related diseases.

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