The gene encoding C/EBP-homologous protein (CHOP), also known as growth arrest and DNA-damageinducible gene 153 (GADD153), is activated by agents that adversely affect the function of the endoplasmic reticulum (ER). Because of the pleiotropic effects of such agents on other cellular processes, the role of ER stress in inducing CHOP gene expression has remained unclear. We find that cells with conditional (temperature-sensitive) defects in protein glycosylation (CHO K12 and BHK tsBN7) induce CHOP when cultured at the nonpermissive temperature. In addition, cells that are defective in initiating the ER stress response, because of overexpression of an exogenous ER chaperone, BiP/GRP78, exhibit attenuated inducibility of CHOP. Surprisingly, attenuated induction of CHOP was also noted in BiP-overexpressing cells treated with methyl methanesulfonate, an agent thought to activate CHOP by causing DNA damage. The roles of DNA damage and growth arrest in the induction of CHOP were therefore reexamined. Induction of growth arrest by culture to confluence or treatment with the enzymatic inhibitor N-(phosphonacetyl)-L-aspartate did not induce CHOP. Furthermore, both a DNA-damage-causing nucleoside analog (5-hydroxymethyl-2-deoxyuridine) and UV light alone did not induce CHOP. These results suggest that CHOP is more responsive to ER stress than to growth arrest or DNA damage and indicate a potential role for CHOP in linking stress in the ER to alterations in gene expression.

The distribution of newly synthesized proteolipid protein (PLP, 23 kdaltons) and myelin basic proteins (MBPs, 14-21.5 kdaltons) was determined in microsomal and myelin fractions prepared from the brainstems oi 10-30 d-old rats sacrificed at different times after an intracranial injection of 3~S-methionine. Labeled MBPs were found in the myelin fraction 2 min after the injection, whereas PLP appeared first in the rough microsomal fraction and only after a lag of 30 rain in the myelin fraction. Cell-free translation experiments using purified mRNAs demonstrated that PLP and MBPs are synthesized in bound and free polysomes, respectively. A mechanism involving the cotranslational insertion into the ER membrane and subsequent passage of the polypeptides through the Golgi apparatus is consistent with the lag observed in the appearance of the in vivo-labeled PLP in the myelin membrane. Newly synthesized PLP and MBPs are not proteolytically processed, because the primary translation products synthesized in vitro had the same electrophoretic mobility and N-terminal amino acid sequence as the mature PLP and MBP polypeptides. It was found that crude myelin fractions are highly enriched in mRNAs coding for the MBPs but not in mRNA coding for PLP. This suggests that whereas the bound polysomes synthesizing PLP are largely confined to the cell body, free polysomes synthesizing MBPs are concentrated in oligodendrocyte processes involved in myelination, which explains the immediate incorporation of MBPs into the developing myelin sheath.The myelin sheath has long been recognized as a rich source of plasma membranes with a relatively simple biochemical composition. In the central nervous system the sheath is derived from the plasma membrane of the oligodendrocyte (c.f. reference 44), a cell that during myelinogenesis greatly expands its surface while developing cytoplasmic processes that wrap around neighboring axons. Compact myelin is formed when the oligodendrocyte cytoplasm is extruded from the myelinating process, thus allowing for a close apposition of adjacent plasma membrane surfaces. In the resulting periodic multilamellar structure, apposed cytoplasmic aspects of the oligodendrocyte plasma membrane form what, in thin-section electron microscopy, is recognized as a major dense line, whereas the extracellular membrane faces form a thinner intraperiod line.The major integral membrane protein of CNS myelin is the myelin proteolipid protein (PLP, or lipophilin, Mr 23 kdaltons) (21, 23) a proteolipid apoprotein that comprises -50% of the total protein (19) and can only be released from the membrane bilayer by treatment with strong detergents or certain organic solvents (23). Myelin also contains a comparable amount of one or a set of peripheral membrane proteins that are easily extracted by acids or high salt treatment, and are known as myelin basic proteins (MBPs), (c.f. reference 11). In the mouse, four different MBP polypeptides have been identified (4). The two major ones (18.5 kdaltons and 14 kdaltons), which we...

Urothelium covers the inner surfaces of the renal pelvis, ureter, bladder and prostatic urethra. Although morphologically indistinguishable, the urothelia in these anatomic locations differ in their embryonic origin and lineages of cellular differentiation, as reflected in their different uroplakin content, expandability during micturition and susceptibility to chemical carcinogens. Previously thought to be an inert tissue forming a passive barrier between the urine and blood, urothelia have recently been shown to have a secretory activity that actively modifies the urine composition. Urothelial cells express a number of ion channels, receptors and ligands, enabling them to receive and send signals and communicate with adjoining cells and their broader environment. The urothelial surface bears specific receptors that not only allow uropathogenic E. coli to attach to and invade into the bladder mucosa, but also provide a route by which the bacteria ascend via the ureters to the kidney to cause pyelonephritis. Genetic ablation of one or more uroplakin genes in mice causes severe retrograde vesicoureteral reflux, hydronephrosis and renal failure, conditions that mirror certain human congenital diseases. Clearly, abnormalities of the lower urinary tract can impact on the upper tract, and vice versa, through the urothelial connection. In this review, we highlight recent advances in the field of urothelial biology by focusing on the uroplakins, a group of urothelium-specific and differentiation-dependent integral membrane proteins. We discuss these proteins’ biochemistry, structure, assembly, intracellular trafficking and their emerging roles in urothelial biology, function and pathological processes. We also call attention to important areas where greater investigative efforts are warranted.

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The apical surface of mammalian urothelium is covered by 16-nm protein particles packed hexagonally to form 2D crystals of asymmetric unit membranes (AUM) that contribute to the remarkable permeability barrier function of the urinary bladder. We have shown previously that bovine AUMs contain four major integral membrane proteins, i.e., uroplakins Ia, Ib, II, and IIIa, and that UPIa and Ib (both tetraspanins) form heterodimers with UPII and IIIa, respectively. Using a panel of antibodies recognizing different conformational states of uroplakins, we demonstrate that the UPIa-dependent, furin-mediated cleavage of the prosequence of UPII leads to global conformational changes in mature UPII and that UPIb also induces conformational changes in its partner UPIIIa. We further demonstrate that tetraspanins CD9, CD81, and CD82 can stabilize their partner protein CD4. These results indicate that tetraspanin uroplakins, and some other tetraspanin proteins, can induce conformational changes leading to the ER-exit, stabilization, and cell surface expression of their associated, single-transmembrane-domained partner proteins and thus can function as "maturation-facilitators." We propose a model of AUM assembly in which conformational changes in integral membrane proteins induced by uroplakin interactions, differentiation-dependent glycosylation, and the removal of the prosequence of UPII play roles in regulating the assembly of uroplakins to form AUM.

The ER's capacity to process proteins is limited, and stress caused by accumulation of unfolded and misfolded proteins (ER stress) contributes to human disease. ER stress elicits the unfolded protein response (UPR), whose components attenuate protein synthesis, increase folding capacity, and enhance misfolded protein degradation. Here, we report that P58 IPK /DNAJC3, a UPR-responsive gene previously implicated in translational control, encodes a cytosolic cochaperone that associates with the ER protein translocation channel Sec61. P58 IPK recruits HSP70 chaperones to the cytosolic face of Sec61 and can be crosslinked to proteins entering the ER that are delayed at the translocon. Proteasome-mediated cytosolic degradation of translocating proteins delayed at Sec61 is cochaperone dependent. In P58 IPKÀ/À mice, cells with a high secretory burden are markedly compromised in their ability to cope with ER stress. Thus, P58 IPK is a key mediator of cotranslocational ER protein degradation, and this process likely contributes to ER homeostasis in stressed cells.