When the protein folding capacity of the endoplasmic reticulum (ER) is challenged, the unfolded protein response (UPR) maintains ER homeostasis by regulating protein synthesis and enhancing expression of resident ER proteins that facilitate protein maturation and degradation. Here, we report that enforced expression of XBP1(S), the active form of the XBP1 transcription factor generated by UPR-mediated splicing of XBP1 mRNA, is sufficient to induce synthesis of phosphatidylcholine, the primary phospholipid of the ER membrane. Cells overexpressing XBP1(S) exhibit elevated levels of membrane phospholipids, increased surface area and volume of rough ER, and enhanced activity of the cytidine diphosphocholine pathway of phosphatidylcholine biosynthesis. These data suggest that XBP1(S) links the mammalian UPR to phospholipid biosynthesis and ER biogenesis.

Development of the expansive endoplasmic reticulum (ER) present in specialized secretory cell types requires X-box-binding protein-1 (Xbp-1). Enforced expression of XBP-1(S), a transcriptional activator generated by unfolded protein responsemediated splicing of Xbp-1 mRNA, is sufficient to induce proliferation of rough ER. We previously showed that XBP-1(S)-induced ER biogenesis in fibroblasts correlates with increased production of phosphatidylcholine (PtdCho), the primary phospholipid of the ER membrane, and enhanced activities of the choline cytidylyltransferase (CCT) and cholinephosphotransferase enzymes in the cytidine diphosphocholine (CDP-choline) pathway of PtdCho biosynthesis. Here, we report that the level and synthesis of CCT, the rate-limiting enzyme in the CDPcholine pathway, is elevated in fibroblasts overexpressing XBP-1(S). Furthermore, overexpression experiments demonstrated that raising the activity of CCT, but not cholinephosphotransferase, is sufficient to augment PtdCho biosynthesis in fibroblasts, indicating that XBP-1(S) increases the output of the CDP-choline pathway primarily via its effects on CCT. Finally, fibroblasts overexpressing CCT up-regulated PtdCho synthesis to a level similar to that in XBP-1(S)-transduced cells but exhibited only a small increase in rough ER and no induction of secretory pathway genes. The more robust XBP-1(S)-induced ER expansion was accompanied by induction of a wide array of genes encoding proteins that function either in the ER or at other steps in the secretory pathway. We propose that XBP-1(S) regulates ER abundance by coordinately increasing the supply of membrane phospholipids and ER proteins, the key ingredients for ER biogenesis. The endoplasmic reticulum (ER)3 is a multifunctional organelle responsible for the folding and assembly of all proteins targeted to the secretory pathway (1). As such, the ER can adapt to accommodate an increased load of nascent polypeptides. For example, when B-lymphocytes differentiate into antibody-secreting plasma cells, an elaborate network of rough ER develops to facilitate immunoglobulin production (2-4). Likewise, the rough ER is highly developed in other specialized secretory cell types such as pancreatic acinar cells that secrete copious amounts of digestive enzymes (5). In contrast, the ER is sparse in non-secretory cells, such as reticulocytes (6). ER abundance, therefore, is regulated according to the demands on the secretory pathway. However, the mechanisms that regulate ER biogenesis are incompletely defined (7).A key regulator of ER homeostasis is the unfolded protein response (UPR) pathway, a complex signaling system emanating from the ER membrane (8). When the protein folding capacity of the ER is challenged, the UPR relieves the resulting stress by repressing translation, increasing expression of ER chaperones and folding enzymes, and enhancing ER-associated degradation (8). In addition, recent studies have uncovered a connection between the UPR and ER abundance (9, 10). The UPR-regulated transcription fa...

A link exists between endoplasmic reticulum (ER) biogenesis and the unfolded protein response (UPR), a complex set of signaling mechanisms triggered by increased demands on the protein folding capacity of the ER. The UPR transcriptional activator X-box binding protein 1 (XBP1) regulates the expression of proteins that function throughout the secretory pathway and is necessary for development of an expansive ER network. We previously demonstrated that overexpression of XBP1(S), the active form of XBP1 generated by UPR-mediated splicing of Xbp1 mRNA, augments the activity of the cytidine diphosphocholine (CDP-choline) pathway for biosynthesis of phosphatidylcholine (PtdCho) and induces ER biogenesis. Another UPR transcriptional activator, activating transcription factor 6α (ATF6α), primarily regulates expression of ER resident proteins involved in the maturation and degradation of ER client proteins. Here, we demonstrate that enforced expression of a constitutively active form of ATF6α drives ER expansion and can do so in the absence of XBP1(S). Overexpression of active ATF6α induces PtdCho biosynthesis and modulates the CDP-choline pathway differently than does enforced expression of XBP1(S). These data indicate that ATF6α and XBP1(S) have the ability to regulate lipid biosynthesis and ER expansion by mechanisms that are at least partially distinct. These studies reveal further complexity in the potential relationships between UPR pathways, lipid production and ER biogenesis.

During the export of flavivirus particles through the secretory pathway, a viral envelope glycoprotein, prM, is cleaved by the proprotein convertase furin; this cleavage is required for the subsequent rearrangement of receptor-binding E glycoprotein and for virus infectivity. Similar to many furin substrates, prM in vector-borne flaviviruses contains basic residues at positions P1, P2, and P4 proximal to the cleavage site; in addition, a number of charged residues are found at position P3 and between positions P5 and P13 that are conserved for each flavivirus antigenic complex. The influence of additional charged residues on pr-M cleavage and virus replication was investigated by replacing the 13-amino-acid, cleavage-proximal region of a dengue virus (strain 16681) with those of tick-borne encephalitis virus (TBEV), yellow fever virus (YFV), and Japanese encephalitis virus (JEV) and by comparing the resultant chimeric viruses generated from RNA-transfected mosquito cells. Among the three chimeric viruses, cleavage of prM was enhanced to a larger extent in JEVpr/16681 than in YFVpr/16681 but was slightly reduced in TBEVpr/16681. Unexpectedly, JEVpr/16681 exhibited decreased focus size, reduced peak titer, and depressed replication in C6/36, PS, and Vero cell lines. The reduction of JEVpr/16681 multiplication correlated with delayed export of infectious virions out of infected cells but not with changes in specific infectivity. Binding of JEVpr/16681 to immobilized heparin and the heparin-inhibitable infection of cells were not altered. Thus, diverse pr-M junction-proximal sequences of flaviviruses differentially influence pr-M cleavage when tested in a dengue virus prM background. More importantly, greatly enhanced prM cleavability adversely affects dengue virus export while exerting a minimal effect on infectivity. Because extensive changes of charged residues at the pr-M junction, as in JEVpr/16681, were not observed among a large number of dengue virus isolates, these results provide a possible mechanism by which the sequence conservation of the pr-M junction of dengue virus is maintained in nature.The genus Flavivirus within the family Flaviviridae comprises about 73 enveloped RNA viruses that are transmitted by either mosquitoes or ticks or without a known vector (11). For these viruses, a single-stranded RNA genome of about 11 kb encodes a polyprotein, which is cleaved by cellular and viral enzymes into three structural proteins (C, prM/M, and E) and seven nonstructural proteins (54). Virions consist of two envelope proteins, E and prM/M, and an internal C protein, which binds genomic RNA. Differences in antigenicities of E allow the subdivision of flaviviruses into eight antigenic complexes and a number of unclassified viruses, which include the prototype yellow fever virus (YFV) (12). More recent assignments based on nucleotide sequence variations of the nonstructural gene NS5 generally agree with antigenic classifications (49).The assembly of flaviviruses in the endoplasmic reticulum is followed by modif...