Incorporation of tubulin subunits into dimers requires GTP hydrolysis

1993 ◽  
Vol 106 (2) ◽  
pp. 627-632 ◽  
Author(s):  
A. Fontalba ◽  
R. Paciucci ◽  
J. Avila ◽  
J.C. Zabala
Keyword(s):  
Protein Folding ◽  
Atp Hydrolysis ◽  
In Vitro Translation ◽  
Magnesium Ions ◽  
Gtp Hydrolysis ◽  
Step Process ◽  
Tubulin Isotypes ◽  
Cytoplasmic Proteins ◽  
Formation Of Complexes

A toroid multisubunit complex of 800–900 kDa has been implicated in assisting protein folding of at least two cytoplasmic proteins, actin and tubulin. This process is dependent on the presence of magnesium ions and ATP hydrolysis. In vitro translation of cDNAs encoding different alpha- and beta-tubulin isotypes also gives rise to the formation of complexes of about 300 kDa. These complexes have been functionally implicated in the incorporation of tubulin monomers within the tubulin heterodimer. This work shows that, in addition to ATP hydrolysis, the incorporation of newly synthesized tubulin subunits into functional heterodimers requires GTP hydrolysis in the presence of magnesium ions. A two-step process is suggested, a first ATP-dependent step in which the 900 kDa complexes are implicated in a similar way to the step taking place in actin folding, and a second GTP-dependent step in which the 300 kDa complexes are involved in the assembly of the heterodimer.

scholarly journals Proteolytic cleavage of haptoglobin occurs in a subcompartment of the endoplasmic reticulum: evidence from membrane fusion in vitro.

1993 ◽  
Vol 123 (2) ◽  
pp. 285-291 ◽  
Author(s):  
M Wassler ◽  
E Fries
Keyword(s):  
Liver Homogenate ◽  
Rat Hepatocytes ◽  
Translation Product ◽  
Alkaline Ph ◽  
Ph Optimum ◽  
Gtp Hydrolysis ◽  
Cytoplasmic Proteins ◽  
Gamma S ◽  
Stimulation Of

The primary translation product of haptoglobin mRNA is a 45-kD polypeptide which is proteolytically cleaved shortly after its synthesis. Previous studies have indicated that the cleavage of this proform of haptoglobin occurs in the ER. In an attempt to characterize the cleaving enzyme, we found that upon incubation of microsomes from rat hepatocytes pulse labeled with [35S]methionine, little cleavage of labeled prohaptoglobin occurred. In contrast, when cells whose cytoplasmic proteins had been released by saponin treatment were incubated, 30-40% of the prohaptoglobin was cleaved. The addition of GTP caused a twofold stimulation, which was abolished by the nonhydrolyzable analog GTP gamma S. With a homogenate of the cells, the addition of GTP resulted in a fourfold stimulation of the degree of cleavage--from 15 to 60%. Differential centrifugation revealed that most of the cleaving activity resided in membranes sedimenting similarly to mitochondria and to a small fraction of the ER. These rapidly sedimenting membranes were therefore prepared from a rat liver homogenate. Upon treatment with high salt, light membranes were released which, when incubated with microsomes of pulse-labeled hepatocytes in the presence of detergent (and in the absence of GTP), induced specific cleavage of prohaptoglobin. The cleaving enzyme had an alkaline pH optimum indicating that it was not of lysosomal origin. These results suggest that cleavage of prohaptoglobin occurs in a subcompartment of the ER. Apparently, the connection between this compartment and the bulk of the ER is broken upon saponin treatment or homogenization but can be reestablished through a process requiring GTP hydrolysis.

scholarly journals In vitro insertion of the 22-kD peroxisomal membrane protein into isolated rat liver peroxisomes.

1993 ◽  
Vol 123 (6) ◽  
pp. 1717-1725 ◽  
Author(s):  
P Diestelkötter ◽  
W W Just
Keyword(s):  
Membrane Protein ◽  
Rat Liver ◽  
Atp Hydrolysis ◽  
Firefly Luciferase ◽  
In Vitro Translation ◽  
Membrane Insertion ◽  
Peroxisomal Membrane ◽  
Permeabilized Cells ◽  
Peroxisomal Membrane Protein

The membrane insertion of the 22-kD integral peroxisomal membrane protein (PMP 22) was studied in a system in which peroxisomes isolated from rat liver were incubated with the [35S]methionine-labeled in vitro translation product of PMP 22 mRNA. Membrane insertion of PMP 22 was demonstrated by protease treatment of peroxisomes in the absence and presence of detergent. Approximately 35% of total in vitro translated PMP 22 became protease resistant after a 1-h incubation at 26 degrees C. Import was dependent on time and temperature, did not require ATP or GTP and was not inhibited by N-ethylmaleimide treatment of neither the soluble components of the translation mixture nor of the isolated peroxisomes. In contrast to these results it was recently shown that the import of the peroxisomal marker, firefly luciferase, into peroxisomes of permeabilized cells was dependent on ATP hydrolysis and was blocked by N-ethylmaleimide pretreatment of the cytosol-depleted cells (Rapp et al., 1993; Wendland and Subramani, 1993). Therefore, the present data suggest that insertion of PMP 22 into the peroxisomal membrane and translocation of firefly luciferase into peroxisomes follow distinct mechanisms. At low temperature binding of PMP 22 to the peroxisomal membrane was not influenced whereas insertion was strongly inhibited. Pretreatment of peroxisomes with subtilisin reduced binding to a low level and completely abolished insertion. Therefore it is suggested that binding is prerequisite to insertion and that insertion may be mediated by a proteinaceous receptor.

scholarly journals Synonymous codon substitutions affect ribosome traffic and protein folding during in vitro translation

FEBS Letters ◽  
1999 ◽  
Vol 462 (3) ◽  
pp. 387-391 ◽  
Author(s):  
Anton A Komar ◽  
Thierry Lesnik ◽  
Claude Reiss
Keyword(s):  
Protein Folding ◽  
Synonymous Codon ◽  
In Vitro Translation

scholarly journals SecY alterations that impair membrane protein folding and generate a membrane stress

2007 ◽  
Vol 176 (3) ◽  
pp. 307-317 ◽  
Author(s):  
Nobuyuki Shimohata ◽  
Shushi Nagamori ◽  
Yoshinori Akiyama ◽  
H. Ronald Kaback ◽  
Koreaki Ito
Keyword(s):  
Protein Folding ◽  
Membrane Proteins ◽  
Membrane Protein ◽  
Signal Recognition Particle ◽  
Membrane Vesicles ◽  
In Vitro Translation ◽  
Membrane Protein Folding ◽  
Protein Biogenesis ◽  
Simple Reduction

We report on a class of Escherichia coli SecY mutants that impair membrane protein folding. The mutants also up-regulate the Cpx/σE stress response pathways. Similar stress induction was also observed in response to a YidC defect in membrane protein biogenesis but not in response to the signal recognition particle–targeting defect or in response to a simple reduction in the abundance of the translocon. Together with the previous contention that the Cpx system senses a protein abnormality not only at periplasmic and outer membrane locations but also at the plasma membrane, abnormal states of membrane proteins are postulated to be generated in these secY mutants. In support of this notion, in vitro translation, membrane integration, and folding of LacY reveal that mutant membrane vesicles allow the insertion of LacY but not subsequent folding into a normal conformation recognizable by conformation-specific antibodies. The results demonstrate that normal SecY function is required for the folding of membrane proteins after their insertion into the translocon.

scholarly journals GTP Is Required for Iron-Sulfur Cluster Biogenesis in Mitochondria

2007 ◽  
Vol 283 (3) ◽  
pp. 1362-1371 ◽  
Author(s):  
Boominathan Amutha ◽  
Donna M. Gordon ◽  
Yajuan Gu ◽  
Elise R. Lyver ◽  
Andrew Dancis ◽  
...  
Keyword(s):  
Atp Hydrolysis ◽  
Dependent Manner ◽  
Gtp Hydrolysis ◽  
Iron Sulfur Cluster ◽  
Isolated Mitochondria ◽  
Iron Sulfur ◽  
The Matrix

Iron-sulfur (Fe-S) cluster biogenesis in mitochondria is an essential process and is conserved from yeast to humans. Several proteins with Fe-S cluster cofactors reside in mitochondria, including aconitase [4Fe-4S] and ferredoxin [2Fe-2S]. We found that mitochondria isolated from wild-type yeast contain a pool of apoaconitase and machinery capable of forming new clusters and inserting them into this endogenous apoprotein pool. These observations allowed us to develop assays to assess the role of nucleotides (GTP and ATP) in cluster biogenesis in mitochondria. We show that Fe-S cluster biogenesis in isolated mitochondria is enhanced by the addition of GTP and ATP. Hydrolysis of both GTP and ATP is necessary, and the addition of ATP cannot circumvent processes that require GTP hydrolysis. Both in vivo and in vitro experiments suggest that GTP must enter into the matrix to exert its effects on cluster biogenesis. Upon import into isolated mitochondria, purified apoferredoxin can also be used as a substrate by the Fe-S cluster machinery in a GTP-dependent manner. GTP is likely required for a common step involved in the cluster biogenesis of aconitase and ferredoxin. To our knowledge this is the first report demonstrating a role of GTP in mitochondrial Fe-S cluster biogenesis.

scholarly journals Requirement of different mitochondrial targeting sequences of the yeast mitochondrial transcription factor Mtf1p when synthesized in alternative translation systems

2004 ◽  
Vol 383 (2) ◽  
pp. 383-391 ◽  
Author(s):  
Tapan K. BISWAS ◽  
Godfrey S. GETZ
Keyword(s):  
Transcription Factor ◽  
Membrane Potential ◽  
Atp Hydrolysis ◽  
Full Length ◽  
In Vitro Translation ◽  
Translation System ◽  
Mitochondrial Transcription Factor ◽  
Reticulocyte Lysate ◽  
Translation Systems

Mitochondrial (mt) translocation of the nuclearly encoded mt transcription factor Mtf1p appears to occur independent of a cleavable presequence, mt receptor, mt membrane potential or ATP [Biswas and Getz (2002) J. Biol. Chem. 277, 45704–45714]. To understand further the import strategy of Mtf1p, we investigated the import of the wild-type and N-terminal-truncated Mtf1p mutants synthesized in two different in vitro translation systems. These Mtf1p derivatives were generated either in the RRL (rabbit reticulocyte lysate) or in the WGE (wheat germ extract) translation system. Under the in vitro import conditions, the RRL-synthesized full-length Mtf1p but not the N-terminal-truncated Mtf1p product was efficiently imported into mitochondria, suggesting that the N-terminal sequence is important for its import. On the other hand, when these Mtf1p products were generated in the WGE system, surprisingly, the N-terminal-truncated products, but not the full-length protein, were effectively translocated into mitochondria. Despite these differences between the translation systems, in both cases, import occurs at a low temperature and has no requirement for a trypsin-sensitive mt receptor, mt membrane potential or ATP hydrolysis. Together, these observations suggest that, in the presence of certain cytoplasmic factors (derived from either RRL or WGE), Mtf1p is capable of using alternative import signals present in different regions of the protein. This appears to be the first example of usage of different targeting sequences for the transport of a single mt protein into the mt matrix.

Analysis of the Mechanism of Microtubule Dynamic Instability Using a UV Microbeam to Sever Elongating Microtubules

1988 ◽  
Vol 46 ◽  
pp. 122-123
Author(s):  
R.A Walker ◽  
S. Inoue ◽  
E.D. Salmon
Keyword(s):  
Dynamic Instability ◽  
Microtubule Dynamic ◽  
Gtp Hydrolysis ◽  
Abrupt Transition ◽  
Microtubule Associated Proteins ◽  
Asymmetrical Structure ◽  
Associated Proteins

Microtubules polymerized in vitro from tubulin purified free of microtubule-associated proteins exhibit dynamic instability (1,2,3). Free microtubule ends exist in persistent phases of elongation or rapid shortening with infrequent, but, abrupt transitions between these phases. The abrupt transition from elongation to rapid shortening is termed catastrophe and the abrupt transition from rapid shortening to elongation is termed rescue. A microtubule is an asymmetrical structure. The plus end grows faster than the minus end. The frequency of catastrophe of the plus end is somewhat greater than the minus end, while the frequency of rescue of the plus end in much lower than for the minus end (4).The mechanism of catastrophe is controversial, but for both the plus and minus microtubule ends, catastrophe is thought to be dependent on GTP hydrolysis. Microtubule elongation occurs by the association of tubulin-GTP subunits to the growing end. Sometime after incorporation into an elongating microtubule end, the GTP is hydrolyzed to GDP, yielding a core of tubulin-GDP capped by tubulin-GTP (“GTP-cap”).

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