An Improved Cell Fractionation Procedure for the Preparation of Rat Liver Membrane-Bound Ribosomes

Adelman, Mark R.; Blobel, Günter; Sabatini, David D.

doi:10.1083/jcb.56.1.191

Cited by 150 publications

(67 citation statements)

References 29 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Gradients were unloaded in 1-ml fractions, and fractions containing ribosomal subunits were pooled as judged by the A 260 profile of each gradient (22). To remove salt and concentrate the ribosomal subunits, samples were diluted with 1 volume of RBB and sedimented (100,000 rpm, 1 h, 4°C, TLA-100.4 rotor, Beckman).…”

Section: Methodsmentioning

confidence: 99%

The α and β Subunit of the Nascent Polypeptide-associated Complex Have Distinct Functions

Beatrix

Sakai

Wiedmann

2000

Journal of Biological Chemistry

View full text Add to dashboard Cite

Nascent polypeptide-associated complex (NAC) is probably the first cytosolic protein to contact nascent polypeptide chains emerging from ribosomes. In this way NAC prevents inappropriate interactions with other factors. Eventually other factors involved in targeting and folding, like the Signal Recognition Particle or cytosolic chaperones, must gain access to the nascent chain. All NAC preparations to date consist of two copurifying polypeptides. Here we rigorously show that these two polypeptides, termed ␣-and ␤NAC, form a very stable complex in vivo and in vitro and that a functional complex can be reconstituted from the individual subunits. A dissection of the contributions of the individual subunits to NACs function revealed that both subunits are in direct contact with nascent polypeptide chains on the ribosome and that both contribute to the prevention of inappropriate interactions. However, ␤NAC alone directly binds to the ribosome and is sufficient to prevent ribosome binding to the endoplasmic reticulum membrane. Nascent polypeptide-associated complex (NAC) 1 is a very abundant cytosolic protein, which is involved in cotranslational targeting of polypeptides to the endoplasmic reticulum (ER) membrane. The intracellular NAC concentration varies only slightly in different tissues, ranging from 3 to 10 M (1). NAC is highly conserved among eukaryotes, but up to now no functional prokaryotic homolog has been described. The importance of NACs in vivo function is emphasized by early embryonically lethal phenotypes of NAC mutants in mice and fruit flies (2, 3). Recently it was also shown that yeast NAC, although not essential for growth (4), is involved in the import of proteins into mitochondria (5, 6).NAC was originally identified as a ribosome-associated protein when we set out to probe the molecular environment of growing polypeptides on the ribosome using a photo-cross-linking approach (7). With this technique we were able to show that NAC can interact with regions of nascent chains as close as 17 amino acids to the peptidyl transferase center. Additionally, it was shown by protease protection that NAC functions as a dissociable wall of a ribosomal tunnel through which the growing polypeptide emerges (8). Cycles of binding and releasing NAC expose the polypeptide to the cytosol in "quantal units" rather than amino acid by amino acid. We have proposed that exposing the polypeptide chain in functional units contributes to fidelity in cotranslational processes such as targeting and folding (9). Depleting NAC from translating ribosomes led to at least two inappropriate interactions. First, the signal recognition particle (SRP), which interacts with signal peptides of nascent chains on ribosomes during targeting to the ER membrane, could be cross-linked to nascent chains lacking signal peptides (7). Second, in the absence of NAC ribosomes translating non-secretory polypeptides inappropriately interacted with translocation sites on ER membranes and the signal-less chains were even translocated, although with l...

show abstract

Section: Methodsmentioning

confidence: 99%

The α and β Subunit of the Nascent Polypeptide-associated Complex Have Distinct Functions

Beatrix

Sakai

Wiedmann

2000

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…(b) It was also demonstrated that, while ribosomes not containing nascent chains can be bound to specific receptors on the ER membranes, such ribosomes are removed from the membranes when microsomes are incubated in media of high salt concentration (2,3,22). On the other hand, ribosomes containing long nascent polypeptides can only be detached from microsomal membranes by treatment with puromycin, which releases the nascent polypeptide chain from the ribosome (2,3), or by destructive methods that unfold the ribosomal subunits (189).…”

Section: Role Of Cotranslational Insertion Signals In Determining Thementioning

confidence: 99%

“…On the other hand, ribosomes containing long nascent polypeptides can only be detached from microsomal membranes by treatment with puromycin, which releases the nascent polypeptide chain from the ribosome (2,3), or by destructive methods that unfold the ribosomal subunits (189). Furthermore, it was shown that as the aminoterminal portion of the nascent chain emerges from the ribosome it enters into a relationship with the membrane that protects the extraribosomal segment of the nascent chain from attack by added proteases, unless detergents are added to dissolve the membranes (185).…”

Section: Role Of Cotranslational Insertion Signals In Determining Thementioning

confidence: 99%

Mechanisms for the incorporation of proteins in membranes and organelles.

Sabatini¹,

Kreibich²,

Morimoto³

et al. 1982

The Journal of Cell Biology

869

281

View full text Add to dashboard Cite

“…The carbon-oxygen bond lengths in the carboxyl group are approximately equal, corresponding to a carboxylate form. The nature of lactim-lactam tautomerism in bilirubin is contro versial [7][8][9][10][11][12][13][14][15][16][17][18][19][20][21][22][23][24][25] . In this study the values of carbon nitrogen and carbon-oxygen bond lengths in the terminal rings, the experi mental evidence in these rings of a hydrogen bound to the nitrogen atom, and the large value observed for the C4 C5-C6 bond angle-as a consequence of H • • H repulsion in the oxodipyrromethene portion (see Fig.…”

mentioning

confidence: 99%

Protein transfer across microsomal membranes reassembled from separated membrane components

Warren

Dobberstein

1978

Nature

113

View full text Add to dashboard Cite

An Improved Cell Fractionation Procedure for the Preparation of Rat Liver Membrane-Bound Ribosomes

Cited by 150 publications

References 29 publications

The α and β Subunit of the Nascent Polypeptide-associated Complex Have Distinct Functions

The α and β Subunit of the Nascent Polypeptide-associated Complex Have Distinct Functions

Mechanisms for the incorporation of proteins in membranes and organelles.

Protein transfer across microsomal membranes reassembled from separated membrane components

Contact Info

Product

Resources

About