The Role of the Leader Sequence Coding Region in Expression and Assembly of Bacteriorhodopsin

Abstract

Bacterio-opsin is made as a precursor in Halobacterium halobium, which has 13 additional residues at the amino terminus. The codons for these residues have been proposed to form a hairpin structure in the mRNA and play a role in ribosome binding; the leader peptide sequence also has been proposed to have a role in membrane insertion of bacteriorhodopsin (BR). We have made mutations in the bop gene region coding for the leader sequence and expressed the mutant genes in an H. halobium mutant lacking wild-type BR. The leader sequence coding region was found to be important for the stability of the mRNA and for its efficient translation. Single base substitutions in this region that did not affect the amino acid sequence caused significant reductions in protein expression. Deletion of the leader region resulted in unstable mRNA and almost no BR production. Introduction of a new ribosome-binding sequence within the coding region of the mature protein restored mRNA stability and some protein expression. Protein made without the leader peptide was properly assembled in the membrane.

Expression of a functionally active human hepatic UDP‐glucuronosyltransferase (UGT1A6) lacking the N‐terminal signal sequence in the endoplasmic reticulum

“…Xu et al [33] recently demonstrated that bacteriorhodopsin made without a 13 residue leader peptide was properly assembled in the membrane. However, deletion of the leader region resulted in an unstable m R N A and almost no bacteriorhodopsin production.…”

Ceramide in Lipid Particles Enhances Heparan Sulfate Proteoglycan and Low Density Lipoprotein Receptor-related Protein-mediated Uptake by Macrophages

“…However, J774 macrophages internalize and degrade both matrix-retained and non-retained SMase-aggregated LDL, which are mediated partially by LRP (32). The size of SMaseaggregated LDL (ϳ100 nm) would be too small to elicit a phagocytic response (19,58). Ö örni et al observed that SMasetreated aggregated and fused LDL bound to human aortic proteoglycans more tightly in the affinity column than native LDL (20,54).…”

“…Xu and Tabas (19) showed that in the presence of a very small amount of SMase (10 milliunits/ml), the endocytosis of LDL was enhanced and led to an increase in LDL degradation and cholesteryl ester accumulation in J774 and mouse peritoneal macrophages. SMase-treated LDL has been shown to be bound and degraded at higher rates than control LDL in a variety of cell lines, including HepG2 cells (56).…”

“…Treatment of LDL particles with SMase from Bacillus cereus has been shown to induce both aggregation and fusion of the particles, which depend on the accumulation of CER within the particles (18 -20). Hydrolysis of LDL by SMase facilitated their uptake by macrophages and induced cholesteryl ester accumulation (foam cell formation) (19,21). The formation of ceramide from SM could thus represent a critical step in atherosclerosis.…”

Acute systemic inflammation up-regulates secretory sphingomyelinase in vivo : A possible link between inflammatory cytokines and atherogenesis

“…First, inflammation plays an important role in atherogenesis (13)(14)(15)(16)(17), and, given the evidence cited above, ASM-mediated ceramide signaling may be responsible for certain inflammatory responses in the developing lesion, such as smooth muscle cell proliferation (25) and apoptosis of macrophages and smooth muscle cells (26,27). Second, one of the products of the ASM gene-secretory SMase (SSMase; see below)-has been linked directly to extracellular subendothelial lipoprotein aggregation, an event that promotes both retention of lipoproteins in the arterial wall and macrophage foam cell formation (25,(28)(29)(30)(31)(32)(33)(34)(35)(36)(37)(38)(39).…”