Abstract. Two COOH terminally truncated variants of ribophorin I (RI), a type I transmembrane glycoprotein of 583 amino acids that is segregated to the rough portions of the ER and is associated with the proteintranslocating apparatus of this organelle, were expressed in permanent HeLa cell transformants . Both variants, one membrane anchored but lacking part of the cytoplasmic domain (RI,67) and the other consisting of the luminal 332 N112-terminal amino acids (RI332), were retained intracellularly but, in contrast to the endogenous long lived, full length ribophorin I (tv2 = 25 h), were rapidly degraded (ti/2 < 50 min) by a nonlysosomal mechanism . The absence of a measurable lag phase in the degradation of both truncated ribophorins indicates that their turnover begins in the ER itself. The degradation of R1467 was monophasic (t 2 = 50 min) but the rate of degradation of R1332 molecules increased about threefold -50 min after their synthesis. Seveal pieces of evidence suggest that the increase in degradative rate is the consequence of the transport of 81332 molecules that are not degraded during the first phase to a second degradative compartment . Thus, IKE any subcellular organelle, the ER contains proteins that permanently reside within it and function in its various biosynthetic and metabolic activities. How ever, it also contains proteins with other subcellular destinations that transit through the ER after they are inserted into its membrane or translocated into its lumen during the course oftheir synthesis on membrane-bound ribosomes (for review see Sabatini and Àdesnik, 1989). Polypeptides synthesized in the ER not only undergo various co-and posttranslational modifications within the organelle, but are also folded and in some cases oligomerized to form multisubunit proteins within the ER itself (for review see Rose and Doms, 1988;Hurtley and Helenius, 1989), apparently assisted by resident proteins which prevent the egress ofincompletely assembled products (Bole et al., 1986; deSilva et al., 1990;Siegel and Walter, 1989). when added immediately after labeling, ionophores that inhibit vesicular flow out of the ER, such as carbonyl cyanide m-chlorophenylhydrazone (CCCP) and monensin, suppressed the second phase of degradation of 81332 . On the other hand, when CCCP was added after the second phase of degradation of 81332 was initiated, the degradation was unaffected. Moreover, in cells treated with brefeldin A the degradation of 81332 became monophasic, and took place with a half-life intermediate between those of the two normal phases. These results point to the existence of two subcellular compartments where abnormal ER proteins can be degraded . One is the ER itself and the second is a nonlysosomal pre-Golgi compartment to which ER proteins are transported by vesicular flow. A survey of the effects of a variety of other ionophores and protease inhibitors on the turnover of 81332 revealed that metalloproteases are involved in both phases of the turnover and that the maintenance of a high Ca 2+ concen...

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Abstract. Ribophorins I and II are type I transmembrane glycoproteins of the ER that are segregated to the rough domains of this organelle. Both ribophorins appear to be part of the translocation apparatus for nascent polypeptides that is associated with membranebound ribosomes and participate in the formation of a proteinaceous network within the ER membrane that also includes other components of the translocation apparatus. The ribophorins are both highly stable proteins that lack O-linked sugars but each contains one high mannose N-linked oligosaccharide that remains endo H sensitive throughout their lifetimes.We have previously shown (Tsao, Y. S., N. E. Ivessa, M. Adesnik, D. D. Sabatini, and G. Kreibich. 1992. J. Cell Biol. 116:57-67) that a COOHterminally truncated variant of ribophorin I that contains only the first 332 amino acids of the luminal domain (RI3a2), when synthesized in permanent transformants of HeLa cells, undergoes a rapid degradation with biphasic kinetics in the ER itself and in a second, as yet unidentified nonlysosomal pre-Golgi compartment. We now show that in cells treated with brefeldin A (BFA) RI332 molecules undergo rapid O-glycosylation in a multistep process that involves the sequential addition of N-acetylgalactosamine, galactose, and terminal sialic acid residues. Addition of O-linked sugars affected all newly synthesized RI332 molecules and was completed soon after synthesis with a half time of about 10 rain. In the same cells, intact ribophorins I and II also underwent O-linked glycosylation in the presence of BFA, but these molecules were modified only during a short time period immediately after their synthesis was completed, and the modification affected only a fraction of the newly synthesized polypeptides. More important, these molecules synthesized before the addition of BFA were not modified by O-glycosylation. The same is true for ribophorin I when overexpressed in HeLa cells although it is significantly less stable than the native polypeptide in control cells. We, therefore, conclude that soon after their synthesis, ribophorins lose their susceptibility to the relocated Golgi enzymes that effect the O-glycosylation, most likely as a consequence of a conformational change in the ribophorins that occurs during their maturation, although it cannot be excluded that rapid integration of these molecules into a supramolecular complex in the ER membrane leads to their inaccessibility to these enzymes. IBOPHORINS I and II are two well characterized, highly stable ER resident glycoproteins that have a type I (N, luminal; C, cytoplasmic) transmembrane disposition and bear high mannose oligosaccharides in their luminal segments (Rosenfeld et al., 1984;Harnik-Ort et al., 1987;Crimaudo et al., 1987). These proteins are segregated to the rough domains of the ER (Kreibich et al., 1978a, b; Macantonio et al., 1984;Amar-Costesec et al., 1984) and

Dihydrofolate reductase (DHFR) system is used to amplify the product gene to multiple copies in Chinese Hamster Ovary (CHO) cells for generating cell lines which produce the recombinant protein at high levels. The physiological changes accompanying the transformation of the non-protein secreting host cells to a high producing cell line is not well characterized. We performed transcriptome analysis on CHO cells undergoing the selection and amplification processes. A host CHO cell line was transfected with a vector containing genes encoding the mouse DHFR (mDHFR) and a recombinant human IgG (hIgG). Clones were isolated following selection and subcloned following amplification. Control cells were transfected with a control plasmid which did not have the hIgG genes. Although methotrexate (MTX) amplification increased the transcript level of the mDHFR gene significantly, its effect on both hIgG heavy and light chain genes was more modest. The subclones appeared to retain the transcriptome signatures of their parental clones, however, their productivity varied among those derived from the same clone. The transcript levels of hIgG transgenes of all subclones fall in a narrower range than the product titer, alluding to the role of many functional attributes, other than transgene transcript, on productivity. We cross examined functional class enrichment during selection and amplification as well as between high and low producers and discerned common features among them. We hypothesize that the role of amplification is not merely increasing transcript levels, but also enriching survivors which have developed the cellular machinery for secreting proteins, leading to an increased frequency of isolating high-producing clones. We put forward the possibility of assembling a hyper-productivity gene set through comparative transcriptome analysis of a wide range of samples.

Sendai virus induced liposome leakage has been studied by using liposomes containing a self-quenching fluorescent dye, calcein. The liposomes used in this study were prepared by a freeze and thaw method and were composed of phosphatidylcholine, phosphatidylserine, and phosphatidylethanolamine (1:2.60:1.48 molar ratio) as well as various amounts of gangliosides and cholesterol. The leakage rate was calculated from the fluorescence increment as the entrapped calcein leaked out of the liposomal compartment and was diluted into the media. It was shown that the target liposome leakage was virus dose dependent. Trypsin-treated Sendai virus in which the F protein had been quantitatively removed did not induce liposome leakage, indicating that the leakage was a direct result of F-protein interaction with the target bilayer membrane. The activation energy of this process was approximately 12 kcal/mol below 17 degrees C and approximately 25 kcal/mol above 17 degrees C. Gangliosides GM1, GD1a, and GT1b could serve as viral receptor under appropriate conditions. Liposome leakage showed a bell-shaped curve dependence on the concentration of ganglioside in the liposomes. No leakage was observed if the ganglioside content was too low or too high. Inclusion of cholesterol in the liposome bilayer suppressed the leakage rate of liposomes containing GD1a. It is speculated that the liposome leakage is a consequence of fusion between Sendai virus and liposomes.