scholarly journals Biosynthesis of the dystonia-associated AAA+ ATPase torsinA at the endoplasmic reticulum

2006 ◽  
Vol 401 (2) ◽  
pp. 607-612 ◽  
Author(s):  
Anna C. Callan ◽  
Sandra Bunning ◽  
Owen T. Jones ◽  
Stephen High ◽  
Eileithyia Swanton
Keyword(s):  
Endoplasmic Reticulum ◽  
Membrane Protein ◽  
Transmembrane Domain ◽  
Signal Sequence ◽  
Integral Membrane Protein ◽  
Signal Peptidase ◽  
Aaa Atpase ◽  
C Terminus ◽  
Membrane Spanning ◽  
Aaa Atpases

TorsinA is a widely expressed AAA+ (ATPases associated with various cellular activities) ATPase of unknown function. Previous studies have described torsinA as a type II protein with a cleavable signal sequence, a single membrane spanning domain, and its C-terminus located in the ER (endoplasmic reticulum) lumen. However, in the present study we show that torsinA is not in fact an integral membrane protein. Instead we find that the mature protein associates peripherally with the ER membrane, most likely through an interaction with an integral membrane protein. Consistent with this model, we provide evidence that the signal peptidase complex cleaves the signal sequence of torsinA, and we show that the region previously suggested to form a transmembrane domain is translocated into the lumen of the ER. The finding that torsinA is a peripheral, and not an integral membrane protein as previously thought, has important implications for understanding the function of this novel ATPase.

scholarly journals Conversion of a class II integral membrane protein into a soluble and efficiently secreted protein: multiple intracellular and extracellular oligomeric and conformational forms.

1990 ◽  
Vol 110 (4) ◽  
pp. 999-1011 ◽  
Author(s):  
R G Paterson ◽  
R A Lamb
Keyword(s):  
Monoclonal Antibodies ◽  
Membrane Protein ◽  
Signal Sequence ◽  
Integral Membrane Protein ◽  
Signal Peptidase ◽  
Protein Fusion ◽  
Native Form ◽  
Hydrophobic Domain ◽  
External Domain ◽  
Signal Anchor

The NH2 terminus of the F1 subunit of the paramyxovirus SV5 fusion protein (fusion related external domain; FRED) is a hydrophobic domain that is implicated as being involved in mediating membrane fusion. We have examined the ability of the FRED to function as a combined signal/anchor domain by substituting it for the natural NH2-terminal signal/anchor domain of a model type II integral membrane protein: the hybrid protein (NAF) was expressed in eukaryotic cells. The FRED was shown to act as a signal sequence, targeting NAF to the lumen of the ER, by the fact that NAF acquired N-linked carbohydrate chains. Alkali fractionation of microsomes indicated that NAF is a soluble protein in the lumen of the ER, and the results of NH2-terminal sequence analysis showed that the FRED is cleaved at a site predicted to be recognized by signal peptidase. NAF was found to be efficiently secreted (t1/2 approximately 90 min) from the cell. By using a combination of sedimentation velocity centrifugation and immunoprecipitation assays using polyclonal and conformation-specific monoclonal antibodies it was found that extracellular NAF consisted of a mixture of monomers, disulfide-linked dimers, and tetramers. The majority of the extracellular NAF molecules were not reactive with the conformation-specific monoclonal antibodies, suggesting they were not folded in a native form and that only the NAF tetramers had matured to a native conformation such that they exhibited NA activity. The available data indicate that NAF is transported intracellularly in multiple oligomeric and conformational forms.

scholarly journals Transient translocation of the cytoplasmic (endo) domain of a type I membrane glycoprotein into cellular membranes.

1993 ◽  
Vol 120 (4) ◽  
pp. 877-883 ◽  
Author(s):  
N Liu ◽  
D T Brown
Keyword(s):  
Amino Acids ◽  
Membrane Protein ◽  
Signal Sequence ◽  
Attachment Site ◽  
Integral Membrane Protein ◽  
Carboxyl Terminus ◽  
Type I ◽  
E2 Glycoprotein ◽  
Typical Type ◽  
Membrane Spanning

The E2 glycoprotein of the alphavirus Sindbis is a typical type I membrane protein with a single membrane spanning domain and a cytoplasmic tail (endo domain) containing 33 amino acids. The carboxyl terminal domain of the tail has been implicated as (a) attachment site for nucleocapsid protein, and (b) signal sequence for integration of the other alpha-virus membrane proteins 6K and E1. These two functions require that the carboxyl terminus be exposed in the cell cytoplasm (a) and exposed in the lumen of the endoplasmic reticulum (b). We have investigated the orientation of this glycoprotein domain with respect to cell membranes by substituting a tyrosine for the normally occurring serine, four amino acids upstream of the carboxyl terminus. Using radioiodination of this tyrosine as an indication of the exposure of the glycoprotein tail, we have provided evidence that this domain is initially translocated into a membrane and is returned to the cytoplasm after export from the ER. This is the first demonstration of such a transient translocation of a single domain of an integral membrane protein and this rearrangement explains some important aspects of alphavirus assembly.

scholarly journals The human polycystin-2 protein represents an integral membrane protein with six membrane-spanning domains and intracellular N- and C-termini

2010 ◽  
Vol 433 (2) ◽  
pp. 285-294 ◽  
Author(s):  
Helen Hoffmeister ◽  
Anna-Rachel Gallagher ◽  
Anne Rascle ◽  
Ralph Witzgall
Keyword(s):  
Endoplasmic Reticulum ◽  
Experimental Evidence ◽  
Transient Receptor Potential ◽  
Receptor Potential ◽  
Integral Membrane Protein ◽  
Protein Product ◽  
C Terminus ◽  
Autosomal Dominant Polycystic Kidney ◽  
Transient Receptor ◽  
Membrane Spanning

PKD2 is one of the two genes mutated in ADPKD (autosomal-dominant polycystic kidney disease). The protein product of PKD2, polycystin-2, functions as a non-selective cation channel in the endoplasmic reticulum and possibly at the plasma membrane. Hydrophobicity plots and its assignment to the TRP (transient receptor potential) family of cation channels suggest that polycystin-2 contains six transmembrane domains and that both the N- and C-termini extend into the cytoplasm. However, no experimental evidence for this model has so far been provided. To determine the orientation of the different loops of polycystin-2, we truncated polycystin-2 within the predicted loops 1–5 and tagged the constructs at the C-terminus with an HA (haemagglutinin) epitope. After transient expression and selective membrane permeabilization, immunofluorescence staining for the HA epitope revealed that loops 1, 3 and 5 extend into the lumen of the endoplasmic reticulum or the extracellular space, whereas loops 2 and 4 extend into the cytoplasm. This approach also confirmed the cytoplasmic orientation of the N- and C-termini of polycystin-2. In accordance with the immunofluorescence data, protease protection assays from microsomal preparations yielded protected fragments when polycystin-2 was truncated in loops 1, 3 and 5, whereas no protected fragments could be detected when polycystin-2 was truncated in loops 2 and 4. The results of the present study therefore provide the first experimental evidence for the topological orientation of polycystin-2.

scholarly journals Borrelia burgdorferi BBA74, a Periplasmic Protein Associated with the Outer Membrane, Lacks Porin-Like Properties

2006 ◽  
Vol 189 (5) ◽  
pp. 2063-2068 ◽  
Author(s):  
Vishwaroop Mulay ◽  
Melissa J. Caimano ◽  
Dionysios Liveris ◽  
Daniel C. Desrosiers ◽  
Justin D. Radolf ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Borrelia Burgdorferi ◽  
Outer Membrane ◽  
Membrane Vesicle ◽  
Integral Membrane Protein ◽  
Proteinase K ◽  
Signal Peptidase ◽  
Signal Peptidase I ◽  
Membrane Spanning ◽  
Α Helix

ABSTRACT The outer membrane of Borrelia burgdorferi, the causative agent of Lyme disease, contains very few integral membrane proteins, in contrast to other gram-negative bacteria. BBA74, a Borrelia burgdorferi plasmid-encoded protein, was proposed to be an integral outer membrane protein with putative porin function and designated as a 28-kDa outer membrane-spanning porin (Oms28). In this study, the biophysical properties of BBA74 and its subcellular localization were investigated. BBA74 is posttranslationally modified by signal peptidase I cleavage to a mature 25-kDa protein. The secondary structure of BBA74 as determined by circular dichroism spectroscopy consists of at least 78% α-helix with little β-sheet structure. BBA74 in intact B. burgdorferi cells was insensitive to proteinase K digestion, and indirect immunofluorescence microscopy showed that BBA74 was not exposed on the cell surface. Triton X-114 extraction of outer membrane vesicle preparations indicated that BBA74 is not an integral membrane protein. Taken together, the data indicate that BBA74 is a periplasmic, outer membrane-associated protein that lacks properties typically associated with porins.

scholarly journals Subcellular Localization and Topology of the p7 Polypeptide of Hepatitis C Virus

2002 ◽  
Vol 76 (8) ◽  
pp. 3720-3730 ◽  
Author(s):  
Séverine Carrère-Kremer ◽  
Claire Montpellier-Pala ◽  
Laurence Cocquerel ◽  
Czeslaw Wychowski ◽  
François Penin ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Subcellular Localization ◽  
Secretory Pathway ◽  
Transmembrane Domain ◽  
Signal Sequence ◽  
C Terminus ◽  
Membrane Spanning ◽  
Polytopic Membrane Protein

ABSTRACT Although biological and biochemical data have been accumulated on most hepatitis C virus proteins, the structure and function of the 63-amino-acid p7 polypeptide of this virus have never been investigated. In this work, sequence analyses predicted that p7 contains two transmembrane passages connected by a short hydrophilic segment. The C-terminal transmembrane domain of p7 was predicted to function as a signal sequence, which was confirmed experimentally by analyzing the translocation of a reporter glycoprotein fused at its C terminus. The p7 polypeptide was tagged either with the ectodomain of CD4 or with a Myc epitope to study its membrane integration, its subcellular localization, and its topology. Alkaline extraction studies confirmed that p7 is an integral membrane polypeptide. The CD4-p7 chimera was detected by immunofluorescence on the surface of nonpermeabilized cells, indicating that it is exported to the plasma membrane. However, pulse-chase analyses showed that only approximately 20% of endoglycosidase H-resistant CD4-p7 was detected after long chase times, suggesting that a large proportion of p7 stays in an early compartment of the secretory pathway. Finally, by inserting a Myc epitope in several positions of p7 and analyzing the accessibility of this epitope on the plasma membrane of HepG2 cells, we showed that p7 has a double membrane-spanning topology, with both its N and C termini oriented toward the extracellular environment. Altogether, these data indicate that p7 is a polytopic membrane protein that could have a functional role in several compartments of the secretory pathway.

scholarly journals Tail-anchored PEX26 targets peroxisomes via a PEX19-dependent and TRC40-independent class I pathway

2013 ◽  
Vol 200 (5) ◽  
pp. 651-666 ◽  
Author(s):  
Yuichi Yagita ◽  
Takahide Hiromasa ◽  
Yukio Fujiki
Keyword(s):  
Amino Acids ◽  
Endoplasmic Reticulum ◽  
Membrane Protein ◽  
Adenosine Triphosphatase ◽  
Transmembrane Domain ◽  
Peroxisomal Membrane ◽  
Basic Amino Acids ◽  
C Terminus ◽  
Peroxisomal Targeting ◽  
Ta Proteins

Tail-anchored (TA) proteins are anchored into cellular membranes by a single transmembrane domain (TMD) close to the C terminus. Although the targeting of TA proteins to peroxisomes is dependent on PEX19, the mechanistic details of PEX19-dependent targeting and the signal that directs TA proteins to peroxisomes have remained elusive, particularly in mammals. The present study shows that PEX19 formed a complex with the peroxisomal TA protein PEX26 in the cytosol and translocated it directly to peroxisomes by interacting with the peroxisomal membrane protein PEX3. Unlike in yeast, the adenosine triphosphatase TRC40, which delivers TA proteins to the endoplasmic reticulum, was dispensable for the peroxisomal targeting of PEX26. Moreover, the basic amino acids within the luminal domain of PEX26 were essential for binding to PEX19 and thereby for peroxisomal targeting. Finally, our results suggest that a TMD that escapes capture by TRC40 and is followed by a highly basic luminal domain directs TA proteins to peroxisomes via the PEX19-dependent route.

scholarly journals An uncleaved glycosylphosphatidylinositol signal mediates Ca2+-sensitive protein degradation

1996 ◽  
Vol 317 (2) ◽  
pp. 533-540
Author(s):  
Peter C. PAULY ◽  
Claudette KLEIN
Keyword(s):  
Endoplasmic Reticulum ◽  
Transmembrane Domain ◽  
Signal Sequence ◽  
Interleukin 2 ◽  
Chimeric Protein ◽  
Integral Membrane Protein ◽  
Marker Enzymes ◽  
Gpi Anchor ◽  
Percoll Gradients ◽  
Anchor Sequence

Inv-gp80 is a chimeric protein which contains a signal for the attachment of a glycosylphosphatidylinositol (GPI) anchor. When expressed in Dictyostelium discoideum, this protein fails to become GPI anchored and is retained within the cell as an integral membrane protein. We have compared the subcellular localization and degradation of Inv-gp80 with that of its intracellular but soluble counterpart, Inv-gp80sc. Inv-gp80sc lacks the hydrophobic C-terminal 22 amino acids of Inv-gp80. The N-linked oligosaccharides of both Inv-gp80 and Inv-gp80sc remained sensitive to endoglycosidase H, and both proteins co-fractionated with endoplasmic reticulum marker enzymes on Percoll gradients. Under normal conditions, Inv-gp80 displayed a half-life (t½) of 90 min, while Inv-gp80sc displayed a t½ of 120 min. The degradation of both proteins required ATP, was inhibited by tosyl phenylalanylchloromethane (Tos-Phe-CH2Cl) and was insensitive to inhibitors of lysosomal function. While depletion of Ca2+ from the endoplasmic reticulum had no effect on the degradation of Inv-gp80sc, it stimulated the degradation of Inv-gp80. When the GPI anchor signal sequence of Inv-gp80 was replaced with the transmembrane domain of the interleukin-2 receptor, the degradation of the protein was no longer influenced by Ca2+ fluxes. The data suggest that while the GPI anchor sequence of Inv-gp80 does not contain determinants regulating the degradation of the protein under basal conditions, it targets Inv-gp80 for rapid degradation under conditions where Ca2+ is depleted from the endoplasmic reticulum.

scholarly journals The PBN1 Gene of Saccharomyces cerevisiae: An Essential Gene That Is Required for the Post-translational Processing of the Protease B Precursor

Genetics ◽  
1998 ◽  
Vol 149 (3) ◽  
pp. 1277-1292 ◽  
Author(s):  
Rajesh R Naik ◽  
Elizabeth W Jones
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Secretory Pathway ◽  
Transmembrane Domain ◽  
Essential Gene ◽  
Signal Sequence ◽  
Signal Peptidase ◽  
Amino Terminal ◽  
Autocatalytic Cleavage ◽  
Protease B ◽  
Chromosome Iii

Abstract The vacuolar hydrolase protease B in Saccharomyces cerevisiae is synthesized as an inactive precursor (Prb1p). The precursor undergoes post-translational modifications while transiting the secretory pathway. In addition to N- and O -linked glycosylations, four proteolytic cleavages occur during the maturation of Prb1p. Removal of the signal peptide by signal peptidase and the autocatalytic cleavage of the large aminoterminal propeptide occur in the endoplasmic reticulum (ER). Two carboxy-terminal cleavages of the post regions occur in the vacuole: the first cleavage is catalyzed by protease A and the second results from autocatalysis. We have isolated a mutant, pbn1-1, that exhibits a defect in the ER processing of Prb1p. The autocatalytic cleavage of the propeptide from Prb1p does not occur and Prb1p is rapidly degraded in the cytosol. PBN1 was cloned and is identical to YCL052c on chromosome III. PBN1 is an essential gene that encodes a novel protein. Pbn1p is predicted to contain a sub-C-terminal transmembrane domain but no signal sequence. A functional HA epitope-tagged Pbn1p fusion localizes to the ER. Pbn1p is N-glycosylated in its amino-terminal domain, indicating a lumenal orientation despite the lack of a signal sequence. Based on these results, we propose that one of the functions of Pbn1p is to aid in the autocatalytic processing of Prb1p.

scholarly journals A Novel Golgi Membrane Protein Is a Partner of the ARF Exchange Factors Gea1p and Gea2p

2003 ◽  
Vol 14 (6) ◽  
pp. 2357-2371 ◽  
Author(s):  
Sophie Chantalat ◽  
Rëgis Courbeyrette ◽  
Francesca Senic-Matuglia ◽  
Catherine L. Jackson ◽  
Bruno Goud ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Protein Trafficking ◽  
Transmembrane Domain ◽  
Integral Membrane Protein ◽  
Membrane Dynamics ◽  
General Interest ◽  
Single Mutation ◽  
Golgi Membrane ◽  
Nucleotide Exchange

The Sec7 domain guanine nucleotide exchange factors (GEFs) for the GTPase ARF are highly conserved regulators of membrane dynamics and protein trafficking. The interactions of large ARF GEFs with cellular membranes for localization and/or activation are likely to participate in regulated recruitment of ARF and effectors. However, these interactions remain largely unknown. Here we characterize Gmh1p, the first Golgi transmembrane-domain partner of any of the high-molecular-weight ARF-GEFs. Gmh1p is an evolutionarily conserved protein. We demonstrate molecular interaction between the yeast Gmh1p and the large ARF-GEFs Gea1p and Gea2p. This interaction involves a domain of Gea1p and Gea2p that is conserved in the eukaryotic orthologues of the Gea proteins. A single mutation in a conserved amino acid residue of this domain is sufficient to abrogate the interaction, whereas the overexpression of Gmh1p can compensate in vivo defects caused by mutations in this domain. We show that Gmh1p is an integral membrane protein that localizes to the early Golgi in yeast and in human HeLa cells and cycles through the ER. Hence, we propose that Gmh1p acts as a positive Golgi-membrane partner for Gea function. These results are of general interest given the evolutionary conservation of both ARF-GEFs and the Gmh proteins.

scholarly journals The Pichia pastoris PER6 gene product is a peroxisomal integral membrane protein essential for peroxisome biogenesis and has sequence similarity to the Zellweger syndrome protein PAF-1.

1996 ◽  
Vol 16 (5) ◽  
pp. 2527-2536 ◽  
Author(s):  
H R Waterham ◽  
Y de Vries ◽  
K A Russel ◽  
W Xie ◽  
M Veenhuis ◽  
...  
Keyword(s):  
Pichia Pastoris ◽  
Membrane Protein ◽  
Sequence Similarity ◽  
Zellweger Syndrome ◽  
Methylotrophic Yeast ◽  
Integral Membrane Protein ◽  
Biochemical Properties ◽  
Podospora Anserina ◽  
Peroxisome Biogenesis ◽  
Membrane Spanning

We report the cloning of PER6, a gene essential for peroxisome biogenesis in the methylotrophic yeast Pichia pastoris. The PER6 sequence predicts that its product Per6p is a 52-kDa polypeptide with the cysteine-rich C3HC4 motif. Per6p has significant overall sequence similarity with the human peroxisome assembly factor PAF-1, a protein that is defective in certain patients suffering from the peroxisomal disorder Zellweger syndrome, and with car1, a protein required for peroxisome biogenesis and caryogamy in the filamentous fungus Podospora anserina. In addition, the C3HC4 motif and two of the three membrane-spanning segments predicted for Per6p align with the C3HC4 motifs and the two membrane-spanning segments predicted for PAF-1 and car1. Like PAF-1, Per6p is a peroxisomal integral membrane protein. In methanol- or oleic acid-induced cells of per6 mutants, morphologically recognizable peroxisomes are absent. Instead, peroxisomal remnants are observed. In addition, peroxisomal matrix proteins are synthesized but located in the cytosol. The similarities between Per6p and PAF-1 in amino acid sequence and biochemical properties, and between mutants defective in their respective genes, suggest that Per6p is the putative yeast homolog of PAF-1.

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