Antibodies to the Golgi complex and the rough endoplasmic reticulum.

Abstract

Rabbits were immunized with membrane fractions from either the Golgi complex or the rough endoplasmic reticulum (RER) by injection into the popliteal lymph nodes. The antisera were then tested by indirect immunofluorescence on tissue culture cells or frozen, thin sections of tissue . There were many unwanted antibodies to cell components other than the RER or the Golgi complex, and these were removed by suitable absorption steps. These steps were carried out until the pattern of fluorescent labeling was that expected for the Golgi complex or RER. Electron microscopic studies, using immunoperoxidase labeling of normal rat kidney (NRK) cells, showed that the anti-Golgi antibodies labeled the stacks of flattened cisternae that comprise the central feature of the Golgi complex, many of the smooth vesicles around the stacks, and a few coated vesicles . These antibodies were directed, almost entirely, against a single polypeptide with an apparent molecular weight of 135,000. The endoplasmic reticulum (ER) in NRK cells is an extensive, reticular network that pervades the entire cell cytoplasm and includes the nuclear membrane . The anti-RER antibodies labeled this structure alone at the light and electron microscopic levels . They were largely directed against four polypeptides with apparent molecular weights of 29,000, 58,000, 66,000, and 91,000 .Some examples are presented, using immunofluorescence microscopy, where these antibodies have been used to study the Golgi complex and RER under a variety of physiological and experimental conditions . For biochemical studies, these antibodies should prove useful in identifying the origin of isolated membranes, particularly those from organelles such as the Golgi complex, which tend to lose their characteristic morphology during isolation .

Neutralizing Monoclonal Antibodies to Bovine Viral Diarrhoea Virus Bind to the 56K to 58K Glycoprotein

“…6 c) (Brown & Farquar, 1984). This is in agreement with previously reported patterns of staining of the Golgi apparatus with fluorescent antibody or lectin probes (Louvard et al, 1982). Clearly, there is no selective accumulation of BDV glycoprotein in the Golgi apparatus of the infected cell.…”

A non-autophagic pathway for diversion of ER secretory proteins to lysosomes

“…4 C). The findings by immunofiuorescence were very similar with an anti-ER serum that recognizes four ER membrane proteins in NRK cells (16): staining was concentrated in the contents of the dilated ER cisternae (Fig. 4, A and B).…”

Misfolded major histocompatibility complex class I molecules accumulate in an expanded ER-Golgi intermediate compartment.

“…The morphology of the class I-enriched reticulum, as observed in plastic-embedded and positively contrasted material, is reminiscent of the site of assembly and accumulation of virus or unassembled viral glycoproteins observed in the CV1 and CHO cell lines (Kartenbeck et al, 1989;Hobman et al, 1992). An antibody that recognizes four ER membrane proteins and is known to label post-ER derivatives (Louvard et al, 1982;Noda and Farquar, 1992), stains the dense tubules and the vesiculated areas enriched in class I HCs (Fig. 6 A), confirming that the tubulo-vesicular profiles are likely derived from, or are part of the ER.…”

“…The following antibodies were used: mAb W6/32 (Barnstable et al, 1978), recognizing properly folded human class I complexes; mAb HC10 , specific for human class I free heavy chains (HCs); antihuman HC serum (Stam et al, 1986), specific for free human HCs; antimouse HC serum (Machold et al, 1995), recognizing free HCs only; anti-p8 serum (raised in our laboratory, according to Smith et al [1986], against a synthetic peptide representing the most COOH-terminal exon of the H-2K b molecule); anti-human 132m serum (raised against highly purified [~2 m, separated from HLA-A2 and HLA-A28 by gel filtration in acetic acid); rabbit anti-ER serum provided by Dr. D. Louvard (Institut Pasteur, Paris, France) (Louvard et al, 1982); mouse monoclonal anti-PDI (ID3) provided by Dr. S. Fuller (EMBL, Heidelberg, Germany) (Vaux et al, 1990); rabbit anti-bovine cathepsin D provided by Dr. G. Jaureguiberry (IN-SERM U.13, Hopital Claude Bernard, Paris, France) (Bailly et al, 1991); mouse monoclonal anti-ERGIC 53 provided by Dr. H. P. Hauri (University of Basel, Basel, Switzerland) (Schweizer et al, 1988); rabbit polyclonal affinity purified anti-human ubiquitin and mouse monoclonal anti-E1 provided by Dr. A. Schwartz (Washington University, St. Louis, MO) (Schwartz et al, , 1992. To visualize the primary antibodies nondirectly reactive with protein A, rabbit anti-mouse IgG and rabbit antimouse IgM (Nordic Immunochemicals, Tilburg, The Netherlands) were used.…”