scholarly journals Proteolytic processing of the α-subunit of rat endopeptidase-24.18 by furin

1995 ◽  
Vol 309 (2) ◽  
pp. 683-688 ◽  
Author(s):  
P E Milhiet ◽  
S Chevallier ◽  
D Corbeil ◽  
N G Seidah ◽  
P Crine ◽  
...  
Keyword(s):  
Transmembrane Domain ◽  
Consensus Sequence ◽  
Proteolytic Processing ◽  
Alpha Subunit ◽  
Structural Domain ◽  
Α Subunit ◽  
Alpha Subunits ◽  
Golgi Compartment ◽  
Membrane Bound

Endopeptidase-24.18 (EC 3.4.24.18; meprin) is a multisubunit metallopeptidase of the astacin family. It is found in brush-border membranes of rodent kidney and human intestine. The membrane-bound enzyme is composed of alpha/beta dimers. Molecular cloning has shown that both subunits have a similar structural domain organization. Soluble alpha 2 dimers have also been observed in vivo and in transfected cells. The structures of all known alpha-subunits contain, upstream from the transmembrane domain, the sequence RXKR, which corresponds to the RXK/RR consensus sequence for specific cleavage by furin. In order to investigate the involvement of this putative cleavage site in the secretion process of endopeptidase-24,.18 alpha-subunit, we expressed in COS-1 cells rat alpha-subunits in which residues R655 or S656 (within the sequence R652PKRS656) were mutated to valine or leucine respectively. In contrast to the wild-type protein, the alpha R655V and alphaS656L mutants were not secreted in the culture medium. Moreover, when cells expressing the alpha-subunit were infected with a furin-encoding vaccinia virus, immunoblotting showed a shift of the major cell-associated form of endopeptidase-24.18 alpha-subunit from 98 kDa to 85 kDa and an increase in the amounts of secreted alpha-subunit. This shift in molecular mass was not observed with the mutant alpha-subunits. As observed for the 98 kDa species, the 85 kDa cell-associated protein was sensitive to endoglycosidase H treatment, suggesting that the proteolytic cleavage occurred in the endoplasmic reticulum or in an early Golgi compartment. Similar experiments using PACE4 and PC5 instead of furin showed that these enzymes were not able to generate the 85 kDa species. We conclude that furin is most probably the cellular enzyme involved in the proteolysis resulting in secretion of rat endopeptidase-24.18 alpha-subunit.

Interaction of the α subunit of Na,K-ATPase with cofilin

2001 ◽  
Vol 353 (2) ◽  
pp. 377-385 ◽  
Author(s):  
Kyunglim LEE ◽  
Jaehoon JUNG ◽  
Miyoung KIM ◽  
Guido GUIDOTTI
Keyword(s):  
Actin Binding ◽  
Cdna Clones ◽  
Cytoplasmic Loop ◽  
Α Subunit ◽  
Transmembrane Segments ◽  
Yeast Two Hybrid System ◽  
Membrane Bound ◽  
Two Hybrid ◽  
Α1 Subunit

The α1 subunit of rat Na,K-ATPase, composed of 1018 amino acids, is arranged in the membrane so that the middle third of the polypeptide forms a large cytoplasmic loop bordered on both sides by multiple transmembrane segments. To identify proteins that might interact with the large cytoplasmic loop of Na,K-ATPase and potentially affect the function and/or the disposition of the pump in the cell, the yeast two-hybrid system was used to screen a rat skeletal muscle cDNA library. Several cDNA clones were isolated, some of which coded for cofilin, an actin-binding protein. Cofilin was co-immunoprecipitated with the α subunit of Na,K-ATPase from extracts of COS-7 cells transiently transfected with haemagglutinin-epitope-tagged cofilin cDNA as well as from yeast extracts. By means of deletion analysis we showed that the segment of cofilin between residues 45 and 99 is essential for functional association with the large cytoplasmic loop of Na,K-ATPase. Recombinant cofilin was shown to bind to the membrane-bound Na,K-ATPase; the association between the two proteins was demonstrated by confocal microscopy. The increased level of cofilin in transfected COS-7 cells caused an increase in the rate of ouabain-sensitive 86Rb+ uptake, indicating that cofilin elicits, either directly or indirectly, enhanced Na,K-ATPase activity and that the interaction occurs in vivo.

scholarly journals Primary structure of the ovine pituitary follitropin α-subunit

1981 ◽  
Vol 197 (3) ◽  
pp. 535-539 ◽  
Author(s):  
M R Sairam
Keyword(s):  
Sequence Analysis ◽  
Primary Structure ◽  
Alpha Subunit ◽  
Partial Sequence ◽  
Tryptic Peptides ◽  
Α Subunit ◽  
Alpha Subunits ◽  
Entire Structure

All the tryptic peptides of reduced and aminoethylated alpha-subunit of ovine pituitary follitropin were isolated. From their their composition and partial sequence analysis of the N- and C-termini of the oxidized protein and reliance on homology with the sequence of lutropin alpha-subunit, an entire structure for the follitropin alpha-subunit has been proposed. The structure of the alpha-subunits from the two ovine hormones are identical.

scholarly journals Atg15 in Saccharomyces cerevisiae consists of two functionally distinct domains

2021 ◽  
pp. mbc.E20-07-0500
Author(s):  
Eri Hirata ◽  
Kyo Shirai ◽  
Tatsuya Kawaoka ◽  
Kosuke Sato ◽  
Fumito Kodama ◽  
...  
Keyword(s):  
Transmembrane Domain ◽  
Multivesicular Body ◽  
Catalytic Triad ◽  
Double Membrane ◽  
Terminal Domain ◽  
C Terminus ◽  
N Terminus ◽  
Membrane Bound ◽  
Important Residue

Autophagy is a cellular degradation system widely conserved among eukaryotes. During autophagy, cytoplasmic materials fated for degradation are compartmentalized in double membrane–bound organelles called autophagosomes. After fusing with the vacuole, their inner membrane–bound structures are released into the vacuolar lumen to become autophagic bodies and eventually degraded by vacuolar hydrolases. Atg15 is a lipase essential for disintegration of autophagic body membranes and has a transmembrane domain at the N-terminus and a lipase domain at the C-terminus. However, the roles of both domains in vivo are not well understood. In this study, we found that the N-terminal domain alone can travel to the vacuole via the multivesicular body pathway, and that targeting of the C-terminal lipase domain to the vacuole is required for degradation of autophagic bodies. Moreover, we found that the C-terminal domain could disintegrate autophagic bodies when it was transported to the vacuole via the Pho8 pathway instead of the multivesicular body pathway. Finally, we identified H435 as one of the residues composing the putative catalytic triad, and W466 as an important residue for degradation of autophagic bodies. This study may provide a clue to understanding how the C-terminal lipase domain recognizes autophagic bodies to degrade them. [Media: see text] [Media: see text]

scholarly journals Developing in vivo assays for investigation of p75NTR and NRH1 transmembrane domain cleavage events using zebrafish embryos

2020 ◽  
Author(s):  
Tanya Jayne ◽  
Morgan Newman ◽  
Michael Lardelli
Keyword(s):  
Alzheimer’S Disease ◽  
Alzheimer's Disease ◽  
Xenopus Laevis ◽  
Amyloid Beta ◽  
Transmembrane Domain ◽  
Chimeric Protein ◽  
Proteolytic Processing ◽  
Structural Basis ◽  
Secretase Inhibitor

Abstractγ-secretase is an important protease complex responsible for the cleavage of over 100 substrates within their transmembrane domains. γ-secretase acts in Alzheimer’s disease by cleavage of AMYLOID BETA (A4) PRECURSOR PROTEIN to produce aggregation-prone Amyloid beta peptide. Other γ-secretase substrates such as p75NTR are also relevant to Alzheimer’s disease. How γ-secretase cleavage site specificity is determined is still unclear. A previous study using Xenopus laevis to investigate the proteolytic processing of p75NTR and its homolog NRH1 found that transmembrane cleavage of NRH1 was not sensitive to the γ-secretase inhibitor DAPT, suggesting that it is not processed by γ-secretase. To investigate this further, we identified zebrafish orthologues of the genes p75NTR and NRH1 and developed in vivo assays to assess cleavage of the resultant p75NTR and Nrh1 proteins. Our observations from these assays in zebrafish are consistent with the Xenopus laevis study. Inhibition of γ-secretase by DAPT treatment results in accumulation of uncleaved p75NTR substrate, while cleavage of Nrh1 is not affected. This supports that p75NTR is cleaved by γ-secretase while Nrh1 is cleaved by a separate γ-secretase-like activity. We extended our approach by generating a chimeric Nrh1 protein in which the Nrh1 transmembrane domain was replaced by that of p75NTR, in an attempt to determine whether it is the p75NTR TMD that confers susceptibility for γ-secretase cleavage. Our results from analysis of this chimeric protein revealed that the p75NTR transmembrane domain alone is insufficient to confer γ-secretase cleavage susceptibility. This is not completely unexpected, as there is evidence to suggest that other factors are crucial for selection/cleavage by the γ-secretase complex. We have established a system in which we can now attempt to dissect the structural basis for γ-secretase cleavage specificity and evolution.

scholarly journals Organization of the Yeast Golgi Complex into at Least Four Funtionally Distinct Compartments

2000 ◽  
Vol 11 (1) ◽  
pp. 171-182 ◽  
Author(s):  
William T. Brigance ◽  
Charles Barlowe ◽  
Todd R. Graham
Keyword(s):  
Golgi Complex ◽  
Brefeldin A ◽  
Proteolytic Processing ◽  
Golgi Compartment ◽  
Specific Antisera ◽  
Sensitive Factor ◽  
Processing Event ◽  
Golgi Network

Pro-α-factor (pro-αf) is posttranslationally modified in the yeast Golgi complex by the addition of α1,6-, α1,2-, and α1,3-linked mannose to N-linked oligosaccharides and by a Kex2p-initiated proteolytic processing event. Previous work has indicated that the α1,6- and α1,3-mannosylation and Kex2p-dependent processing of pro-αf are initiated in three distinct compartments of the Golgi complex. Here, we present evidence that α1,2-mannosylation of pro-αf is also initiated in a distinct Golgi compartment. Linkage-specific antisera and an endo-α1,6-d-mannanase (endoM) were used to quantitate the amount of each pro-αf intermediate during transport through the Golgi complex. We found that α1,6-, α1,2-, and α1,3-mannose were sequentially added to pro-αf in a temporally ordered manner, and that the intercompartmental transport factor Sec18p/N-ethylmaleimide-sensitive factor was required for each step. The Sec18p dependence implies that a transport event was required between each modification event. In addition, most of the Golgi-modified pro-αf that accumulated in brefeldin A-treated cells received only α1,6-mannosylation as did ∼50% of pro-αf transported to the Golgi in vitro. This further supports the presence of an early Golgi compartment that houses an α1,6-mannosyltransferase but lacks α1,2-mannosyltransferase activity in vivo. We propose that the α1,6-, α1,2-, and α1,3-mannosylation and Kex2p-dependent processing events mark the cis, medial,trans, and trans-Golgi network of the yeast Golgi complex, respectively.

scholarly journals Complex Proteolytic Processing Acts on Delta, a Transmembrane Ligand for Notch, during Drosophila Development

1998 ◽  
Vol 9 (7) ◽  
pp. 1709-1723 ◽  
Author(s):  
Kristin M. Klueg ◽  
Todd R. Parody ◽  
Marc A.T. Muskavitch
Keyword(s):  
Cell Fate ◽  
Cultured Cells ◽  
Cellular Responses ◽  
Proteolytic Processing ◽  
Cell Fate Specification ◽  
A Cell ◽  
Membrane Bound ◽  
Delta Functions ◽  
Intracellular Domains

Delta functions as a cell nonautonomous membrane-bound ligand that binds to Notch, a cell-autonomous receptor, during cell fate specification. Interaction between Delta and Notch leads to signal transduction and elicitation of cellular responses. During our investigations to further understand the biochemical mechanism by which Delta signaling is regulated, we have identified four Delta isoforms inDrosophila embryonic and larval extracts. We have demonstrated that at least one of the smaller isoforms, Delta S, results from proteolysis. Using antibodies to the Delta extracellular and intracellular domains in colocalization experiments, we have found that at least three Delta isoforms exist in vivo, providing the first evidence that multiple forms of Delta exist during development. Finally, we demonstrate that Delta is a transmembrane ligand that can be taken up by Notch-expressing Drosophila cultured cells. Cell culture experiments imply that full-length Delta is taken up by Notch-expressing cells. We present evidence that suggests this uptake occurs by a nonphagocytic mechanism.

scholarly journals Immunolocalization of Kex2 protease identifies a putative late Golgi compartment in the yeast Saccharomyces cerevisiae.

1991 ◽  
Vol 113 (3) ◽  
pp. 527-538 ◽  
Author(s):  
K Redding ◽  
C Holcomb ◽  
R S Fuller
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Secretory Pathway ◽  
Killer Toxin ◽  
Proteolytic Processing ◽  
Yeast Cells ◽  
Yeast Saccharomyces Cerevisiae ◽  
Golgi Compartment ◽  
Membrane Bound ◽  
Gene Structures ◽  
Stage Analysis

The Kex2 protein of the yeast Saccharomyces cerevisiae is a membrane-bound, Ca2(+)-dependent serine protease that cleaves the precursors of the mating pheromone alpha-factor and the M1 killer toxin at pairs of basic residues during their transport through the secretory pathway. To begin to characterize the intracellular locus of Kex2-dependent proteolytic processing, we have examined the subcellular distribution of Kex2 protein in yeast by indirect immunofluorescence. Kex2 protein is located at multiple, discrete sites within wild-type yeast cells (average, 3.0 +/- 1.7/mother cell). Qualitatively similar fluorescence patterns are observed at elevated levels of expression, but no signal is found in cells lacking the KEX2 gene. Structures containing Kex2 protein are not concentrated at a perinuclear location, but are distributed throughout the cytoplasm at all phases of the cell cycle. Kex2-containing structures appear in the bud at an early, premitotic stage. Analysis of conditional secretory (sec) mutants demonstrates that Kex2 protein ordinarily progresses from the ER to the Golgi but is not incorporated into secretory vesicles, consistent with the proposed localization of Kex2 protein to the yeast Golgi complex.

scholarly journals The effect of iloprost on the ADP-ribosylation of Gsα (the α-subunit of Gs)

1989 ◽  
Vol 261 (3) ◽  
pp. 841-845 ◽  
Author(s):  
L Molina y Vedia ◽  
R D Nolan ◽  
E G Lapetina
Keyword(s):  
Adenylate Cyclase ◽  
Cholera Toxin ◽  
Alpha Subunit ◽  
Α Subunit ◽  
Present Evidence ◽  
Adp Ribosylation ◽  
Specific Antisera ◽  
Membrane Bound ◽  
Gs Alpha ◽  
Adp Ribosyltransferase

Treatment of platelets with a prostacyclin analogue, iloprost, decreased the cholera-toxin-induced ADP-ribosylation of membrane-bound Gs alpha (alpha-subunit of G-protein that stimulates adenylate cyclase; 42 kDa protein) and a cytosolic substrate (44 kDa protein) [Molina y Vedia, Reep & Lapetina (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 5899-5902]. This decrease is apparently not correlated with a significant change in the quantity of membrane Gs alpha, as detected by two Gs alpha-specific antisera. This finding contrasts with the suggestion in a previous report [Edwards, MacDermot & Wilkins (1987) Br. J. Pharmacol. 90, 501-510], indicating that iloprost caused a loss of Gs alpha from the membrane. Our evidence points to a modification in the ability of the 42 kDa protein to be ADP-ribosylated by cholera toxin. This modification of Gs alpha might be related to its ADP-ribosylation by endogenous ADP-ribosyltransferase activity. Here we present evidence showing that Gs alpha was ADP-ribosylated in platelets that had been electropermeabilized and incubated with [alpha-32P]NAD+. This endogenous ADP-ribosylation of Gs alpha is inhibited by nicotinamide and stimulated by iloprost.

scholarly journals Localization and targeting of the Saccharomyces cerevisiae Kre2p/Mnt1p alpha 1,2-mannosyltransferase to a medial-Golgi compartment.

1995 ◽  
Vol 131 (4) ◽  
pp. 913-927 ◽  
Author(s):  
M Lussier ◽  
A M Sdicu ◽  
T Ketela ◽  
H Bussey
Keyword(s):  
Membrane Protein ◽  
Transmembrane Domain ◽  
Glycosylation Site ◽  
Dependent Manner ◽  
Reporter Protein ◽  
Golgi Localization ◽  
Golgi Compartment ◽  
Membrane Spanning

The yeast Kre2p/Mnt1p alpha 1,2-mannosyltransferase is a type II membrane protein with a short cytoplasmic amino terminus, a membrane-spanning region, and a large catalytic luminal domain containing one N-glycosylation site. Anti-Kre2p/Mnt1p antibodies identify a 60-kD integral membrane protein that is progressively N-glycosylated in an MNN1-dependent manner. Kre2p/Mnt1p is localized in a Golgi compartment that overlaps with that containing the medial-Golgi mannosyltransferase Mnn1p, and distinct from that including the late Golgi protein Kex1p. To determine which regions of Kre2p/Mnt1p are required for Golgi localization, Kre2p/Mnt1p mutant proteins were assembled by substitution of Kre2p domains with equivalent sequences from the vacuolar proteins DPAP B and Pho8p. Chimeric proteins were tested for correct topology, in vitro and in vivo activity, and were localized intracellularly by indirect immunofluorescence. The results demonstrate that the NH2-terminal cytoplasmic domain is necessary for correct Kre2p Golgi localization whereas, the membrane-spanning and stem domains are dispensable. However, in a test of targeting sufficiency, the presence of the entire Kre2p cytoplasmic tail, plus the transmembrane domain and a 36-amino acid residue luminal stem region was required to localize a Pho8p reporter protein to the yeast Golgi.

Rat skeletal muscle phosphorylase kinase: turnover and control of isozyme levels in culture

1986 ◽  
Vol 250 (3) ◽  
pp. C365-C373 ◽  
Author(s):  
W. J. Salsgiver ◽  
J. C. Lawrence
Keyword(s):  
Skeletal Muscle ◽  
Kinase Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Proteolytic Processing ◽  
Alpha Subunit ◽  
Phosphorylase Kinase ◽  
Ionophore A23187 ◽  
Rat Skeletal Muscle ◽  
Alpha Subunits

The expression of phosphorylase kinase was investigated in rat skeletal muscle cells developing in vitro. The enzyme was immunoprecipitated from cells cultured in the presence of [35S]methionine, and the 35S-labeled alpha-, alpha'-, and beta-subunits of the kinase were resolved by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Fusion of myoblasts into myotubes was associated with marked increases in the amounts of kinase activity and the three 35S-labeled subunits. In 2-wk-old myotubes, the net amount of alpha'-subunit represented less than 20% of the total alpha-subunits (alpha + alpha'); however, alpha'-subunits appeared to be synthesized at least as rapidly as alpha-subunits. That alpha'-subunits were degraded more rapidly was confirmed by pulse-chase experiments, which also indicated that alpha'-subunits were not formed by proteolytic processing of the larger alpha-subunit. Inhibition of the spontaneous contractile activity of the myotubes with lidocaine markedly increased both phosphorylase kinase activity and the amounts of the 35S-labeled subunits. The divalent cation ionophore, A23187, decreased the alpha-subunits by 60%, but did not change levels of the alpha'-subunits. Taken together, the present results indicate that rat myotubes synthesize the two isozymes of phosphorylase kinase, and that levels of both are controlled by differentiation and muscle activity.

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