A novel quality control compartment derived from the endoplasmic reticulum.

Degradation of proteins that, because of improper or suboptimal processing, are retained in the endoplasmic reticulum (ER) involves retrotranslocation to reach the cytosolic ubiquitin-proteasome machinery. We found that substrates of this pathway, the precursor of human asialoglycoprotein receptor H2a and free heavy chains of murine class I major histocompatibility complex (MHC), accumulate in a novel preGolgi compartment that is adjacent to but not overlapping with the centrosome, the Golgi complex, and the ER-to-Golgi intermediate compartment (ERGIC). On its way to degradation, H2a associated increasingly after synthesis with the ER translocon Sec61. Nevertheless, it remained in the secretory pathway upon proteasomal inhibition, suggesting that its retrotranslocation must be tightly coupled to the degradation process. In the presence of proteasomal inhibitors, the ER chaperones calreticulin and calnexin, but not BiP, PDI, or glycoprotein glucosyltransferase, concentrate in the subcellular region of the novel compartment. The "quality control" compartment is possibly a subcompartment of the ER. It depends on microtubules but is insensitive to brefeldin A. We discuss the possibility that it is also the site for concentration and retrotranslocation of proteins that, like the mutant cystic fibrosis transmembrane conductance regulator, are transported to the cytosol, where they form large aggregates, the "aggresomes."

Membrane-bound versus secreted forms of human asialoglycoprotein receptor subunits. Role of a juxtamembrane pentapeptide.

The H2a alternatively spliced variant of the human asialoglycoprotein receptor H2 subunit differs from the H2b variant by an extra pentapeptide, EGHRG, present in the ectodomain next to the membrane-span. This difference causes retention and degradation in the endoplasmic reticulum (ER) of H2a when expressed without the H1 subunit in 3T3 cells. In contrast, a significant portion of singly expressed H2b is Golgi-processed and reaches the cell surface. Using a new specific anti-H2a antibody, we found that in HepG2 cells, H2a is rapidly cleaved to a 35-kDa fragment, comprising the entire ectodomain, most of which is secreted into the medium. The cleavage site for the secreted fragment was located at the lumenal end of the membrane span. No membrane-bound H2a exits the ER, indicating that the pentapeptide is a signal for ER retention and degradation of the membrane form but does not hinder secretion of the cleaved soluble form. H2a does not form a membrane receptor complex with H1 as H2b does. H2a is therefore not a subunit of the receptor but a precursor for a secreted form of the protein; signal peptidase is probably responsible for the cleavage to the soluble fragment. Therefore, the juxtamembrane sequence regulates the function of the transmembrane domain of a type II membrane protein as either a signal-anchor sequence (H2b) or as a cleaved signal sequence, which generates a secreted product (H2a).

Use of Tn5lac to study expression of genes required for production of the antibiotic TA.

The beta-galactosidase activities arising from Tn5lac insertions in several genes required for antibiotic TA production were measured under different growth conditions. In all of the non-TA-producing mutants, the beta-galactosidase specific activity was higher when the cells were grown in nutrient-limited 0.5CTS medium (0.5% Casitone plus alanine, serine, and glucose) than in rich 2CT medium (2% Casitone). One of the mutants, 420, had low beta-galactosidase specific activity in both media. The other seven mutants containing inserts in genes essential for TA production had specific activities of 139 to 367 U/mg of protein in 0.5CTS medium and 11 to 48 U/mg of protein in 2CT medium. The beta-galactosidase specific activities of two strains, 1030 and 420, increased during exponential growth in 0.5CTS medium. The beta-galactosidase specific activities of both strains increased greatly when the cells were grown in the presence of magnesium phosphate, which traps ammonium ions. The Tn5lac insertions in 1030 and 420 were used to screen for mutants with increased levels of transcription. An N-methyl-N'-nitro-N-nitrosoguanidine-induced mutation in 1030 that mapped 17 kb from the omega 1010 insert increased the specific activity of beta-galactosidase 21 times in 2CT medium. The regulatory mutation appears to release the repression caused by 2CT medium. A UV-induced mutation in 420 increased the beta-galactosidase specific activity 1.4 to 2.4 times. Medium conditions that affect the transcription of TA genes are discussed in terms of enhanced antibiotic TA production.

Mutation and mapping of genes involved in production of the antibiotic TA in Myxococcus xanthus.

Transposition of TnV and Tn5lac into Myxococcus xanthus yielded 8,381 kanamycin-resistant mutants that were tested for antibiotic TA production. Twenty-four of the mutants were nonproducers of TA (less than 0.4 ng/ml), and 3 produced a higher level (2.5 micrograms/ml) than the parent strain (1.5 micrograms/ml). For most of the strains, there was 100% cotransduction between kanamycin resistance and the altered TA phenotype. Southern blot analysis of restriction digests of the mutant DNA indicated that the transposons were inserted at different sites on the M. xanthus chromosome. The TA genes were mapped by cotransduction between pairs of mutants following replacement of the initial insert of one of the pair with the tetracycline resistance transposon Tn5-132. Nine of the 13 nonproducers tested were linked over a 36-kb stretch of the chromosome. There was no linkage between one of the overproducers and any of the nonproducers tested.