We have used proteinase K as a probe to detect cytoplasmically and lumenally exposed segments of nascent polypeptides undergoing transport across mammalian microsomal membranes. A series of translocation intermediates consisting of discrete-sized nascent chains was prepared by including microsomal membranes in cell-free translations of mRNAs lacking termination codons. The truncated mRNAs were ...

We have used proteinase K as a probe to detect cytoplasmically and lumenally exposed segments of nascent polypeptides undergoing transport across mammalian microsomal membranes. A series of translocation intermediates consisting of discrete-sized nascent chains was prepared by including microsomal membranes in cell-free translations of mRNAs lacking termination codons. The truncated mRNAs were ...

Correct folding of nascent polypeptide chains within the ER is critical for function, assembly into multi-subunit complexes and trafficking through the exocytic pathway for secretory and cell surface proteins. This process is rather inefficient, and a substantial proportion of nascent polypeptides is rejected by an ER quality control system and targeted for degradation. In some cases, only a mi...

Murine monoclonal subunit-specific antibodies against heterooctameric phosphofructokinase-1 from Saccharomyces cerevisiae exhibiting no cross-reactivity were purified and characterized regarding complex formation with the native enzyme, immunological reactivity to the SDS-denatured subunits originating from native and proteolytically truncated phosphofructokinase, and protection against proteol...

A panel of mutant class II genes have been constructed using site-directed mutagenesis and DNA-mediated gene transfer. Using this technique, Ak beta polypeptides have been altered by substituting one or more Ad beta-specific residues at polymorphic positions in the beta 1 domain. Transfection of M12.C3 B lymphoma cells with most mutant Ak beta* genes results in the expression of Ak beta* Ad alp...

The existence of specialized molecular chaperones that interact directly with ribosomes is well established in microorganisms. Such proteins bind polypeptides exiting the ribosomal tunnel and provide a physical link between translation and protein folding. We report that ribosome-associated molecular chaperones have been maintained throughout eukaryotic evolution, as illustrated by Mpp11, the h...

In eukaryotic cells, most polypeptides destined to become membrane proteins are initially integrated into the membrane of the endoplasmic reticulum (ER) before being sorted to the location at which they function. Integration occurs at sites in the ER membrane termed translocons that are comprised of a specific set of membrane proteins (1, 2). In most cases, proteins are integrated into the bila...

Rough microsomes were incubated in an in vitro amino acid-incorporating system for labeling the nascent polypeptide chains on the membrane-bound ribosomes . Sucrose density gradient analysis showed that ribosomes did not detach from the membranes during incorporation in vitro. Trypsin and chymotrypsin treatment of microsomes at 0 ° led to the detachment of ribosomes from the membranes ; further...

The tmRNA system performs translational surveillance and ribosome rescue in all eubacteria and some eukaryotic organelles. This system intervenes when ribosomes read to the 3' end of an mRNA or pause at internal codons with subsequent mRNA cleavage. A complex of alanyl-tmRNA (which functions as a tRNA and mRNA), SmpB protein, and EF-TucGTP binds stalled ribosomes, the nascent polypeptide is tra...

Rough microsomes were incubated in an in vitro amino acid-incorporating system for labeling the nascent polypeptide chains on the membrane-bound ribosomes . Sucrose density gradient analysis showed that ribosomes did not detach from the membranes during incorporation in vitro. Trypsin and chymotrypsin treatment of microsomes at 0 ° led to the detachment of ribosomes from the membranes ; further...