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R Pfaller

Publications and source records attributed to R Pfaller.

17 recordsLinked to original sources
PubMed1997

Isolation and characterisation of a novel stress-inducible PDI-family gene from Aspergillus niger.

Current strategies to improve the secretion of heterologous proteins in Aspergillus niger include the manipulation of chaperones and foldases specific to the endoplasmic reticulum (ER). A family of ER-specific proteins which share active-site homology wit protein disulfide isomerase (PDI) has been identified from other systems, many of which are inducible by agents which cause malfolding of proteins in the ER. Here we report identification of tigA from Aspergillus niger and erp38 from Neurospora crassa, two novel members of the PDI superfamily of proteins. TIGA and ERp38 show 66% identity at the amino acid level and are putative ER proteins. Both proteins show tandemly linked thiol-oxidoreductase domains followed by a functionally uncharacterised C-terminal domain. The most distal active site in TIGA is created by excision of a 66-bp intron. Although no Unfolded Protein Response elements can be seen in the tigA promoter, sequence homology has identified associated with protein trafficking (ERPTRE) in a gene encoding the related mammalian protein, ERp72, as well as a second motif conserved amongst the glucose-related protein family. Southern and dot blot analysis indicate that the tigA gene is present in single copy. Both the A. niger and N. crassa proteins show homology with a stress-inducible alfalfa, G1. Transcription of tigA is induced 2-3-fold after treatment with tunicamycin, an inhibitor of N-linked glycosylation. Strains overexpressing a heterologous protein show no increased tigA mRNA levels.

Amino Acid Sequence↗
PubMed1995

Assembly/disassembly of the nuclear envelope membrane. Characterization of the membrane-chromatin interaction using partially purified regulatory enzymes.

Assembly and disassembly of the nucleus at mitosis in eukaryotes involves the reversible interaction of chromatin with the nuclear membrane. Previously we have shown that this interaction is regulated by the antagonistic activities of a kinase and a phosphatase. The kinase promotes membrane release while the phosphatase stimulates binding. In this report we describe four steps in the purification of the kinase needed for release of membranes from chromatin. We also show that the release kinase and the mitotic initiation kinase, cdc2, are distinct and are separated from each other during the second purification step. Reconstitution experiments using these two kinases demonstrate that the release kinase and cdc2 kinase work in concert to cause membrane release from chromatin. In phosphorylation experiments, protein targets that are substrates for the regulatory release kinase are identified on the membranes. These phosphorylated proteins ae candidates for regulated proteins mediating membrane-chromatin interaction. Finally, we find that membrane release activity can also be extracted from membranes by high salt treatment, indicating a possible dual localization of this activity.

Animals↗
PubMed1994

Expression of synthetic cDNA sequences encoding human insulin-like growth factor-1 (IGF-1) in the mammary gland of transgenic rabbits.

We have developed an expression system where foreign proteins are synthesized specifically in the mammary gland of transgenic rabbits and secreted into the milk. Regulatory elements were isolated from the bovine alpha S1-casein-encoding gene and combined with a synthetic DNA coding for human IGF-1 and for [Gln58]IGF-1, an IGF-1 analogue. The resulting hybrid DNA constructs were used to generate transgenic rabbits. Females of seven transgenic lines tested were positive for synthesis of IGF-1. Transmission of the transgene to progeny and IGF-1 production in female offspring was observed in all transgenic lines analysed. As expected, expression of transgene mRNA could only be detected in the mammary gland. Production levels of transgenic protein were as high as 1 g IGF-1 per liter rabbit milk. IGF-1, as well as [Gln58]IGF-1, when secreted into rabbit milk, was correctly processed and biologically active. IGF-1 was purified from the milk of transgenic rabbits to a nearly homogenous active form.

Animals↗
PubMed1991

Assembly/disassembly of the nuclear envelope membrane: cell cycle-dependent binding of nuclear membrane vesicles to chromatin in vitro.

Dissociation and association of membranes with chromatin at the beginning and end of mitosis are critical in controlling nuclear dynamics during these stages of the cell cycle. Employing purified membrane and cytosolic fractions from Xenopus eggs, a simple assay was developed for the reversible binding of nuclear membrane vesicles to chromatin. We have shown, using phosphatase and kinase inhibitors, that membrane-chromatin association is regulated by a phosphatase/kinase system. In interphase, the balance in this system favors dephosphorylation, possibly of a membrane receptor, which then mediates chromatin binding. At mitosis the membrane receptor is phosphorylated, causing release of chromatin-bound membrane. Purified MPF kinase does not directly cause membranes to dissociate from chromatin. Rather, binding of membranes to chromatin at mitosis appears to be regulated indirectly by MPF through its action on a phosphatase/kinase system that directly modulates the phosphorylation state of a nuclear membrane component.

Animals↗
PubMed1990

A mitochondrial import receptor for the ADP/ATP carrier.

We have identified a mitochondrial outer membrane protein of 72 kd (MOM72) that exhibits the properties of an import receptor for the ADP/ATP carrier (AAC), the most abundant mitochondrial protein. Monospecific antibodies and Fab fragments against MOM72 selectively inhibit import of AAC at the level of specific binding to the mitochondria. AAC bound to the mitochondrial surface is coprecipitated with antibodies against MOM72 after lysis of mitochondria with detergent. MOM72 thus has a complementary function to that of MOM19, which acts as an import receptor for the majority of mitochondrial proteins studied so far but not for the AAC. The import pathway of the precursor of MOM72 appears to involve MOM19 as receptor.

Animals↗
PubMed1990

Biogenesis of mitochondrial porin: the import pathway.

We review here the present knowledge about the pathway of import and assembly of porin into mitochondria and compare it to those of other mitochondrial proteins. Porin, like all outer mitochondrial membrane proteins studied so far is made as a precursor without a cleavable 'signal' sequence; thus targeting information must reside in the mature sequence. At least part of this information appears to be located at the amino-terminal end of the molecule. Transport into mitochondria can occur post-translationally. In a first step, the porin precursor is specifically recognized on the mitochondrial surface by a protease sensitive receptor. In a second step, porin precursor inserts partially into the outer membrane. This step is mediated by a component of the import machinery common to the import pathways of precursor proteins destined for other mitochondrial subcompartments. Finally, porin is assembled to produce the functional oligomeric form of an integral membrane protein which is characterized by its extreme protease resistance.

Animals↗
PubMed1990

Import of ADP/ATP carrier into mitochondria: two receptors act in parallel.

We have identified the yeast homologue of Neurospora crassa MOM72, the mitochondrial import receptor for the ADP/ATP carrier (AAC), by functional studies and by cDNA sequencing. Mitochondria of a yeast mutant in which the gene for MOM72 was disrupted were impaired in specific binding and import of AAC. Unexpectedly, we found a residual, yet significant import of AAC into mitochondria lacking MOM72 that occurred via the receptor MOM19. We conclude that both MOM72 and MOM19 can direct AAC into mitochondria, albeit with different efficiency. Moreover, the precursor of MOM72 apparently does not require a positively charged sequence at the extreme amino terminus for targeting to mitochondria.

Amino Acid Sequence↗
PubMed1989

MOM19, an import receptor for mitochondrial precursor proteins.

We have identified a 19 kd protein of the mitochondrial outer membrane (MOM19). Monospecific IgG and Fab fragments directed against MOM19 inhibit import of precursor proteins destined for the various mitochondrial subcompartments, including porin, cytochrome c1, Fe/S protein, F0 ATPase subunit 9, and F1 ATPase subunit beta. Inhibition occurs at the level of high affinity binding of precursors to mitochondria. Consistent with previous functional studies that suggested the existence of distinct import sites for ADP/ATP carrier and cytochrome c, we find that import of those precursors is not inhibited. We conclude that MOM19 is identical to, or closely associated with, a specific mitochondrial import receptor.

Animals↗
PubMed1989

Mitochondrial protein import. Bypass of proteinaceous surface receptors can occur with low specificity and efficiency.

Proteolytic degradation of receptor sites on the mitochondrial surface strongly reduces the efficiency of mitochondrial protein import. The remaining residual import still involves basic mechanisms of protein import, including: insertion of precursors into the outer membrane, requirement for ATP and a membrane potential, and translocation through contact sites between both membranes. The import of a chloroplast protein into isolated mitochondria which occurs with a low rate is not inhibited by a protease-pretreatment of mitochondria, indicating that this precursor only follows the bypass pathway. The low efficiency of bypass import suggests that this unspecific import does not disturb the uniqueness of mitochondrial protein composition. We conclude that mitochondrial protein import involves a series of steps in which receptor sites appear to be responsible for the specificity of protein uptake.

Adenosine Triphosphate↗
PubMed1988

Role of ATP in mitochondrial protein import. Conformational alteration of a precursor protein can substitute for ATP requirement.

The role of nucleoside triphosphates (NTPs) in the import of porin into the mitochondrial outer membrane was investigated with two forms of the porin precursor: the in vitro synthesized biosynthetic precursor (bs-porin) and a water-soluble form of porin (ws-porin) obtained by subjecting the membrane-derived porin to an acid-base treatment (exposure to trichloroacetic acid, followed by alkali and rapid neutralization). The import of ws-porin into mitochondria did not require NTPs, whereas the import of bs-porin required NTPs. In other characteristics, such as binding to a specific receptor protein on the mitochondrial surface, two-step insertion into the outer membrane, and formation of specific membrane channels, ws-porin was indistinguishable from bs-porin. Thus, the acid-base treatment applied in the preparation of ws-porin can substitute for the NTP-requiring step in mitochondrial protein import. We conclude that NTPs are required for unfolding mitochondrial precursor proteins ("translocation competent folding").

Adenosine Triphosphate↗
PubMed1988

Import pathways of precursor proteins into mitochondria: multiple receptor sites are followed by a common membrane insertion site.

The precursor of porin, a mitochondrial outer membrane protein, competes for the import of precursors destined for the three other mitochondrial compartments, including the Fe/S protein of the bc1-complex (intermembrane space), the ADP/ATP carrier (inner membrane), subunit 9 of the F0-ATPase (inner membrane), and subunit beta of the F1-ATPase (matrix). Competition occurs at the level of a common site at which precursors are inserted into the outer membrane. Protease-sensitive binding sites, which act before the common insertion site, appear to be responsible for the specificity and selectivity of mitochondrial protein uptake. We suggest that distinct receptor proteins on the mitochondrial surface specifically recognize precursor proteins and transfer them to a general insertion protein component (GIP) in the outer membrane. Beyond GIP, the import pathways diverge, either to the outer membrane or to translocation contact-sites, and then subsequently to the other mitochondrial compartments.

Binding Sites↗
PubMed1987

Mitochondrial porin of Neurospora crassa: cDNA cloning, in vitro expression and import into mitochondria.

cDNA encoding porin of Neurospora crassa, the major protein component of the outer mitochondrial membrane, was isolated and the nucleotide sequence was determined. The deduced protein sequence consists of 283 amino acids (29,979 daltons) and shows sequence homology of around 43% to yeast porin; however, no significant homology to bacterial porins was apparent. According to secondary structure predictions, mitochondrial porin consists mainly of membrane-spanning sided beta-sheets. Porin was efficiently synthesized in vitro from the cDNA; this allowed us to study in detail its import into mitochondria. Thereby, three characteristics of import were defined: (i) import depended on the presence of nucleoside triphosphates; (ii) involvement of a proteinaceous receptor-like component on the surface of the mitochondria was demonstrated; (iii) insertion into the outer membrane was resolved into at least two distinct steps: specific binding to high-affinity sites and subsequent assembly to the mature form.

Amino Acid Sequence↗
PubMed1987

High-affinity binding sites involved in the import of porin into mitochondria.

The specific recognition by mitochondria of the precursor of porin and the insertion into the outer membrane were studied with a radiolabeled water-soluble form of porin derived from the mature protein. High-affinity binding sites had a number of 5-10 pmol/mg mitochondrial protein and a ka of 1-5 X 10(8) M-1. Binding was abolished after trypsin pretreatment of mitochondria indicating that binding sites were of protein-aceous nature. Specifically bound porin could be extracted at alkaline pH but not by high salt and was protected against low concentrations of proteinase K. It could be chased to a highly protease resistant form corresponding to mature porin. High-affinity binding sites could be extracted from mitochondria with detergent and reconstituted in asolectin-ergosterol liposomes. Water-soluble porin competed for the specific binding and import of the precursor of the ADP/ATP carrier, an inner membrane protein. We suggest that (i) binding of precursors to proteinaceous receptors serves as an initial step for recognition, (ii) the receptor for porin may also be involved in the import of precursors of inner membrane proteins, and (iii) interaction with the receptor triggers partial insertion of the precursor into the outer membrane.

Binding Sites↗
PubMed1985

A water-soluble form of porin from the mitochondrial outer membrane of Neurospora crassa. Properties and relationship to the biosynthetic precursor form.

Mitochondrial porin, the outer membrane pore-forming protein, was isolated in the presence of detergents and converted into a water-soluble form. This water-soluble porin existed under nondenaturing conditions as a mixture of dimers and oligomers. The proportion of dimers increased with decreasing porin concentration during conversion. Water-soluble porin inserted spontaneously into artificial bilayers as did detergent-solubilized porin. Whereas the latter form had no specific requirements for the lipid composition of the bilayer, water-soluble porin inserted only into membranes containing a sterol, and only in the presence of very low concentrations of Triton X-100 (0.001% w/v) in the solution bathing the bilayer. The channels formed by water-soluble porin were indistinguishable from those formed by detergent-purified porin with respect to specific conductance and voltage dependence of conductance. Water-soluble porin bound tightly in a saturable fashion to isolated mitochondria. The bound form was readily accessible to added protease, indicating its presence on the mitochondrial surface. The number of binding sites was in the range of 5-10 pmol/mg of mitochondrial protein. Water-soluble porin apparently binds to a site on the assembly pathway of the porin precursor, since mitochondria whose binding sites were saturated with the water-soluble form did not import porin precursor synthesized in a cell-free system.

Bacterial Outer Membrane Proteins↗