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Quality control in the endoplasmic reticulum must discriminate nascent proteins in their folding process from terminally unfolded molecules, selectively degrading the latter. Unassembled Ig-mu and J chains, two glycoproteins with five N-linked glycans and one N-linked glycan, respectively, are degraded by cytosolic proteasomes after a lag from synthesis, during which glycan trimming occurs. Inhibitors of mannosidase I (kifunensine), but not of mannosidase II (swainsonine), prevent the degradation of mu chains. Kifunensine also inhibits J chain dislocation and degradation, without inhibiting secretion of IgM polymers. In contrast, glucosidase inhibitors do not significantly affect the kinetics of mu and J degradation. These results suggest that removal of the terminal mannose from the central branch acts as a timer in dictating the degradation of transport-incompetent, glycosylated Ig subunits in a calnexin-independent way. Kifunensine does not inhibit the degradation of an unglycosylated substrate (lambda Ig light chains) or of chimeric mu chains extended with the transmembrane region of the alpha T cell receptor chain, implying the existence of additional pathways for extracting proteins from the endoplasmic reticulum lumen for proteasomal degradation.

Oxidizing conditions must be maintained in the endoplasmic reticulum (ER) to allow the formation of disulfide bonds in secretory proteins. Here we report the cloning and characterization of a mammalian gene (ERO1-L) that shares extensive homology with the Saccharomyces cerevisiae ERO1 gene, required in yeast for oxidative protein folding. When expressed in mammalian cells, the product of the human ERO1-L gene co-localizes with ER markers and displays Endo-H-sensitive glycans. In isolated microsomes, ERO1-L behaves as a type II integral membrane protein. ERO1-L is able to complement several phenotypic traits of the yeast thermosensitive mutant ero1-1, including temperature and dithiothreitol sensitivity, and intrachain disulfide bond formation in carboxypeptidase Y. ERO1-L is no longer functional when either one of the highly conserved Cys-394 or Cys-397 is mutated. These results strongly suggest that ERO1-L is involved in oxidative ER protein folding in mammalian cells.

IgM secreting cells express little or no membrane IgM. This is not always due to absence of the relevant mRNA. To investigate the synthesis and processing of membrane (micron) and secreted (microseconds) polypeptides in secretory B cells, myeloma cells were transfected either with a plasmid containing an intact mu gene or with one only capable of directing micron (not microseconds) mRNA synthesis. Although myeloma transfectants could make abundant levels of micron mRNA, they did not express IgM on the cell surface. In the myeloma host, micron mRNA is translated some 5‐fold less efficiently than microseconds mRNA. However, this translational control does not totally preclude micron synthesis, indicating post‐translational regulatory events. No difference between micron and microseconds chains could be detected in their rate of assembly with light chains or in their stability, although both types of heavy chain were degraded more rapidly when synthesized in the absence of light chain, or when the hydrophobic nature of the leader sequence was destroyed by site‐directed mutagenesis. However, whereas intracellular microseconds chains in IgM‐secreting plasmacytoma were found to be concentrated in the Golgi, the micron chains were mainly located in the endoplasmic reticulum. Retention in the endoplasmic reticulum is also observed for both micron and microseconds when synthesized in the absence of light chain. We propose that it is the expansion of the endoplasmic reticulum that accompanies B cell to plasma cell differentiation which is in part responsible for the down‐regulation of surface IgM expression. Such a mechanism may also affect the expression of other surface proteins.

The capacity of synthesizing and secreting Ig molecules was studied in 11 patients with B-cell chronic lymphocytic leukemia (B-CLL) whose cells expressed surface IgM, in 3 patients with surface IgG-bearing cells, and in 2 IgM prolymphocyte leukemias (IgM-PLL). Three types of μchains were detected by SDS-polyacrylamide gel electrophoresis analysis of the endogenously labeled molecules isolated by specific immunoprecipitation. Two of them were isolated from the cell lysates and were identified as the membrane μchain and the precursor of the secreted molecules, respectively. The latter also possibly contained precursors of the membrane molecules. The third type of molecule was detected only in the culture medium and was identified as secretory μchain. Not all of the malignant clones possessed the three types of μchains. Only of the IgM-bearing malignant cell clones were capable of secretion, whereas the remaining synthesized the secretory μchains but degraded them intracellularly. Two types of molecules (membrane and secreted) were found in the IgG-bearing CLL cells from three patients. In all of them, secretion was detected. Ultrastructural analysis demonstrated that cells from the secreting clones had the features of more mature lymphocytes than the cells from nonsecreting clones. These features were represented by a developed Golgi apparatus, various types of vesicles (smooth and coated), and strands of the rough endoplasmic reticulum. A certain heterogeneity of the degree of maturation of the cells was observed within these clones. The data are consistent with the hypothesis that CLL clones are heterogeneous and can be distinguished through the different degrees of maturation of their cell components.

We have compared the synthesis and processing of immunoglobulin alpha chains in two murine cell lines, a B cell lymphoma that expresses membrane-bound IgA and a hybridoma that secretes IgA. Results of biosynthetic labeling experiments demonstrated that membrane-bound and secreted alpha chains have two distinct intracellular precursors, of different molecular weights and isoelectric points. RNAs from both of these cell lines direct the synthesis in vitro of two alpha polypeptides of Mr 59,000 and 62,000, the larger one being the precursor for membrane-bound alpha chain and the smaller one being the precursor for secreted alpha chain. These cell lines each contain three RNAs, 1.7, 2.1, and 3.1 kilobases in length, which hybridize with cDNA for the alpha constant region and which are present in different concentrations. Our results suggest that the smallest RNA encodes the secreted alpha chain and one or both of the larger RNAs encode(s) the membrane-bound alpha chain.

Human immunodeficiency virus-1 (HIV-1) Tat, a nuclear transcription factor, has been shown to function extracellularly, implying that some Tat molecules escape nuclear import and are secreted. This raises the question of what regulates, in HIV-1-infected cells, the nuclear targeting of the polypeptide. Here we show that cytosolic components activated by Ca2+ ions are required to reveal the karyophilic properties of Tat: in vitro translated Tat molecules do not associate with isolated nuclei unless preincubated with Ca2+. Moreover, Ca2+ ions induce karyophilicity of chemically synthesized Tat molecules only upon addition of cytosolic extracts. The Ca2+-induced karyophilicity is prevented by inhibitors of either tyrosine kinases (herbimycin A and genistein) or tyrosine phosphatases (vanadate), suggesting the involvement of Ca2+-dependent phosphorylation/dephosphorylation events. In line with these observations, the transcriptional activity of Tat is inhibited by treatment with either vanadate or genistein. The same occurs with Tat mutants lacking either one or both the two tyrosine residues (positions 26 and 47). Hence, Ca2+-dependent tyrosine kinase(s) and phosphatase(s) act on accessory cellular protein(s), which in turn are responsible of Tat karyophilicity.

Immunoglobulin heavy-chain switching is effected by a DNA recombination event that replaces the C mu gene with one of the other heavy-chain constant-region (CH) genes located 3' to the C mu gene. How the specificity of this event is controlled is unknown. However, it has been shown that IgM+ cells capable of switching to specific isotypes have the corresponding unrearranged CH genes in an accessible or active chromatin state, as demonstrated by the fact that these specific CH genes are hypomethylated and are transcriptionally active. We now report that the RNAs transcribed from specific unrearranged CH genes are induced prior to switching under conditions that promote switching to these specific CH genes. For example, we find that bacterial lipopolysaccharide, which induces the IgM+ cell line I.29 mu to switch to IgA, induces transcripts from the germ-line C alpha gene(s) in I.29 mu cells prior to switch recombination. Two preparations of T-cell lymphokines (recombinant interleukin 4 and supernatant from the T-cell line 2.19, which contains interleukins 4 and 5) that promote switching to specific isotypes by lipopolysaccharide-treated spleen cells induce transcripts from the corresponding germ-line CH genes prior to expression of the new isotypes. For example, interleukin 4, which appears to be necessary for switching to IgE in vitro and in vivo, induces within 2 days large increases in germ-line C epsilon transcripts in lipopolysaccharide-treated spleen cells and in I.29 mu cells. The most straightforward interpretation of our data is that these lymphokines direct switching to specific isotypes by activating specific CH genes, making them accessible to the putative switch recombinase.

The cysteine present in the Ig micro chain tailpiece (microtp) prevents the secretion of unpolymerized IgM intermediates and causes their accumulation in the endoplasmic reticulum (ER). In principle, this can be the consequence of actual retention in this organelle or of retrieval from the Golgi. To determine which of the two mechanisms underlies the cysteine-dependent ER localization, we analyze here the post-translational modifications of suitably engineered cathepsin D (CD) molecules. The glycans of this protease are phosphorylated by post-ER phosphotransferases and further modified in the trans-Golgi to generate a mannose 6-phosphate lysosome targeting signal. Only trace amounts of the mutp-tagged CD (CDM&mutpCys) are phosphorylated, unless retention is reversed by exogenous reducing agents or the critical cysteine mutated (CDMmutpSer). In contrast, a KDEL-tagged CD, that is retrieved from the Golgi into the ER, acquires phosphates, though mainly resistant to alkaline phosphatase. Similarly to CDMmutpSer, the few CDMmutpCys molecules that escape retention and acquire phosphates in the cis-Golgi are transported beyond the KDEL retrieval compartment, as indicated by their sensitivity to alkaline phosphatase. These results demonstrate that the thiol-dependent ER localization arises primarily from true retention, without recycling through the Golgi.

CD36 is an integral membrane glycoprotein expressed by several cell types, including endothelial cells of the microvasculature, erythrocytes, platelets, and monocytes. In the monocytic lineage, CD36 is expressed during the late stages of differentiation in the bone marrow, in circulating monocytes, and in some tissue resident macrophages, and it is thought to mediate the phagocytosis of apoptotic cells and the endocytic uptake of modified lipoproteins. Here we analyze the synthesis, processing, and intracellular transport of CD36 in U937 and THP-1, two human cell lines representing different stages of monocytic maturation. In both cell lines, phorbol 12-myristate 13-acetate induces the expression of CD36. A 74-kDa intracellular precursor is first synthesized that has the hallmarks of a resident protein of the endoplasmic reticulum. The precursor protein is later processed into a mature form of 90-105 kDa which is transported to the cell surface. The kinetics of processing differ significantly in U937 and THP-1. These differences are specific for the CD36, as two unrelated proteins (CD11b and CD45R) are processed and transported to the surface at similar rates in the two cell lines. A 33-kDa endoglycosidase H-sensitive glycoprotein specifically associates with the 74-kDa precursor. Coprecipitation of gp33 correlates with slow processing of CD36 precursor, suggesting that gp33 may play a role in regulating the intracellular transport of CD36, during monocyte maturation.

Production of interleukin 1 (IL-1) by leukemic cells was studied in 13 cases of acute myeloid leukemia. Intracytoplasmic immunofluorescence studies showed that the cells invariably contained the cytokine. Endogenous labeling studies demonstrated that acute myeloid leukemia cells produced either only the 33-kDa propeptide or both the propeptide and the 17-kDa mature form of IL-1 beta. The 33-kDa propeptide IL-1 alpha was always produced but was less frequently released. Involvement of IL-1 in leukemic cell growth was investigated using two antibodies specific for IL-1 subtypes, which inhibited spontaneous cell proliferation in the six cases studied. After acid treatment of the cells, a surface receptor for IL-1 could be demonstrated, which mediated 125I-labeled IL-1-specific uptake by leukemic cells. Furthermore, recombinant IL-1 alpha or IL-1 beta induced significant cell proliferation in 10 of 12 cases. The above findings were uncorrelated with the cytologic type (French-American-British classification) of leukemia. Our studies suggest that IL-1 may act as an autocrine growth factor in most cases of acute myeloid leukemia.