| Summary: | 碩士 === 國立中興大學 === 分子生物學研究所 === 98 === Bacterial lipoproteins (Lpp) are proteins N-terminally modified by lipidation and use their lipid moiety for membrane anchorage. The genes encoding NMB1523 and NMB1533, Lpp of Neisseria meningitidis (NM), have previously been cloned for expression in E. coli. However, Western blotting showed that removal of signal peptide caused a failure in NMB1523 expression but not NMB1533, suggesting that these two proteins may have different biosynthetic pathways. In this study, cells of E. coli carrying the cloned genes were fractionated by lysozyme-osmotic shock and different fractions were analyzed by Western blotting. Results indicated that NMB1533 but not NMB1523 was detected in cytoplasm, suggesting that NMB1533 was translocated post-translationally and NMB1523 co-translationally. To elucidate Lpp biosynthesis in NM, subcellular localization of 9 Lpp in log-phase and stationary-phase NM cells was determined by Western blotting. Results of samples prepared by lysozyme-osmotic shock method indicated that NMB1523, NMB1533, NMB0278 (DsbA-1), NMB1468 (Ag473) and NMA1697 were not detected in cytoplasmic fraction, suggesting that these Lpp were translocated co-translationally; in contrast, NMB0294 (DsbA-2), NMB0550 (DsbC), NMB1164 and NMB1946 were detected in cytoplasm fraction, suggesting post-translational translocation of these Lpp. It has previously been shown that in E. coli and P. aeruginosa, an Lpp is retained in the inner membrane if the mature protein contains an Asp at position +2, otherwise it will be located in outer membrane. Since the rules followed by NM for subcellular localization of Lpp remained unclear, distribution of these Lpp were analyzed by Western blotting with membrane fractions prepared by a chemical method. Results showed that all lipoproteins were detectable in both inner and outer membranes. Western blotting was also performed with samples prepared by sucrose gradient centrifugation. Results showed that 1) equal amounts of NMB0278 (DsbA-1), NMB0294 (DsbA-2), NMB0550 (DsbC), NMB1533 and NMB1946 were detected in both inner and outer membranes, 2) majority of NMB1468 (Ag473) and NMB1523 was detected in inner membrane fraction, and 3) NMB1164 and NMA1697 were primarily detected in outer membrane fraction. Examination of the amino acid at position +2 revealed that 1) NMB0278 (DsbA-1) has Asp at + 2, but it is located in both inner and outer membranes in equal amount, and 2) NMB1468 (Ag473) and NMB1523, without a Asp at +2, are primarily distributed in inner membrane. Together these results indicate that Lpp sorting in NM may be governed by rules different from that in E. coli and P. aeurginosa, although the export pathways may be similar to that of E. coli in which most outer membrane and periplasmic proteins are translocated post-translationally, whereas most inner membrane Lpp are translocated co-translationally.
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