Corrections BIOCHEMISTRY. For the article ‘‘RNA degradosomes exist in vivo in Escherichia coli as multicomponent complexes associated with the cytoplasmic membrane via the N-terminal region of ribo- nuclease E’’ by Gunn-Guang Liou, Wann-Neng Jane, Stanley N. Cohen, Na-Sheng Lin, and Sue Lin-Chao, which appeared in number 1, January 2, 2001, of Proc. Natl. Acad. Sci. USA (98, 63–68; First Published December 26, 2000; 10.1073͞ pnas.011535498), the authors note that the y axis label of Fig. 1B ͞ should read ‘‘picomoles OD600.’’ The corresponding text in the Fig. 1 legend on page 64 and the results described on page 66, line 2 from the bottom of the left-hand column, should also be in units of picomole per OD600. www.pnas.org͞cgi͞doi͞10.1073͞pnas.161275298 NEUROBIOLOGY. For the article ‘‘In vivo induction of massive proliferation, directed migration, and differentiation of neural cells in the adult mammalian brain’’ by James Fallon, Steve Reid, Richard Kinyamu, Isaac Opole, Rebecca Opole, Janie Baratta, Murray Korc, Tiffany L. Endo, Alexander Duong, Gemi Nguyen, Masoud Karkehabadhi, Daniel Twardzik, and Sandra Loughlin, which appeared in number 26, December 19, 2000, of Proc. Natl. Acad. Sci. USA (97, 14686–14691), the authors note the follow- ing correction. Dr. Sanjiv Patel’s name and affiliation were omitted from the list of authors. Dr. Patel’s affiliation is St. Joseph’s Hospital and Medical Center, Paterson, NJ 07503. The corrected list of authors is: James Fallon, Steve Reid, Richard Kinyamu, Isaac Opole, Rebecca Opole, Janie Baratta, Murray Korc, Tiffany L. Endo, Alexander Duong, Gemi Nguyen, Ma- soud Karkehabadhi, Daniel Twardzik, Sanjiv Patel, and Sandra Loughlin. www.pnas.org͞cgi͞doi͞10.1073͞pnas.161282198 CORRECTIONS www.pnas.org PNAS ͉ July 3, 2001 ͉ vol. 98 ͉ no. 14 ͉ 8157 Downloaded by guest on September 25, 2021 RNA degradosomes exist in vivo in Escherichia coli as multicomponent complexes associated with the cytoplasmic membrane via the N-terminal region of ribonuclease E Gunn-Guang Liou*†, Wann-Neng Jane‡, Stanley N. Cohen§, Na-Sheng Lin‡, and Sue Lin-Chao*¶ *Institute of Molecular Biology, Academia Sinica, Nankang, Taipei 115, Taiwan; †Institute of Molecular Medicine, College of Medicine, National Taiwan University, Taipei 100, Taiwan; ‡Institute of Botany, Academia Sinica, Nankang, Taipei 115, Taiwan; and §Department of Genetics, Stanford University School of Medicine, Stanford, CA 94305-5120 Contributed by Stanley N. Cohen, November 9, 2000 RNase E isolated from Escherichia coli is contained in a multicom- region that interacts with PNPase and other degradosome ponent ‘‘degradosome’’ complex with other proteins implicated in proteins (15), and that purified RNase E devoid of other RNA decay. Earlier work has shown that the C-terminal region of degradosome components is functionally active in vitro (16–19). RNase E is a scaffold for the binding of degradosome components We set out to investigate the existence of multicomponent and has identified specific RNase E segments necessary for its degradosomes in vivo and also to localize degradosome proteins. interaction with polynucleotide phosphorylase (PNPase), RhlB RNA We report here that degradosomes are present in intact E. coli helicase, and enolase. Here, we report electron microscopy studies cells. We further show that degradosomes, whose composition that use immunogold labeling and freeze–fracture methods to appears to be dynamically regulated, are not distributed evenly show that degradosomes exist in vivo in E. coli as multicomponent throughout the cytoplasm—but instead are associated with the structures that associate with the cytoplasmic membrane via the cytoplasmic membrane via the N-terminal region of RNase E. N-terminal region of RNase E. Whereas PNPase and enolase are present in E. coli in large excess relative to RNase E and therefore Materials and Methods are detected in cells largely as molecules unlinked to the RNase E Bacterial Strains and Plasmids. The following E. coli strains were scaffold, immunogold labeling and biochemical analyses show that Ϫ Ϫ used: HB101 [supE44 hsdS20(rB mB ) recA13 ara-14 proA2 helicase is present in approximately equimolar amounts to RNase lacY1 galK2 rpsL20 xyl-5 mtl-1], BL21(DE3) [hsdS gal(␭cIts857 E at all cell growth stages. Our findings, which establish the ind1 S am7 nin5 lacUV5-T7 gene1)], and BZ99 [his ⌬trpE5 existence and cellular location of RNase E-based degradosomes in mukB106 smbB199 (␭)] (13). HB101 and BL21(DE3) contain vivo in E. coli, also suggest that RNA processing and decay may wild-type rne gene, and BZ99 contains a truncated rne gene that occur at specific sites within cells. encodes amino acids 1 through 602 of the Rne protein. Plasmid pGP1–2 contains the T7 RNA polymerase gene under the immunogold labeling ͉ RhlB RNA helicase ͉ RNA process ͉ RNA ␭ degradation control of a temperature-sensitive bacteriophage repressor (20). Plasmid pRE296 is identical to pRE196 (3) except that the Flag tag was replaced with a His6-Flag tag. Nase E is an essential Escherichia coli ribonuclease that has ͞ Ra key role in the degradation and or processing of both short Degradosome Complex Purifications. RNA degradosomes were and long half-lived RNAs. When purified from E. coli cells, purified as described in ref. 3 by using the Flag-epitope-tagged RNase E is present in a multicomponent ribonucleolytic complex Rne fusion protein and a Flag-monoclonal antibody-conjugated (i.e., the RNA ‘‘degradosome’’) that includes polynucleotide agarose column. phosphorylase (PNPase), the RhlB RNA helicase, enolase, the DnaK chaperonin protein, GroEL, and polynucleotide phos- Antibody Preparation. Polyclonal antibodies were prepared as phate kinase (PPK) (1–5). Specific regions required to bind described (21). Briefly, purified degradosomes were separated certain degradosome proteins have been identified within the by SDS͞PAGE and stained with Coomassie blue. The gel slices C-terminal half of RNase E (6), and a functionally active containing individual bands of RNase E, PNPase, enolase, or minimal degradosome containing only RNase E, PNPase, and RhlB helicase were frozen in liquid nitrogen, dried, and ground helicase has been reconstituted in vitro (7). The view that many, if not most, cellular functions are carried into a powder. The powdered gel was suspended in Freund’s out in vivo by multicomponent macromolecular complexes (i.e., adjuvant and phosphate buffer (pH 7.2) used to immunize cellular machines) rather than by individual freely diffusable rabbits. Degradosome components in protein gels and E. coli proteins has gained wide acceptance in recent years (8). Well cells were detected by using the same antibodies, except that the recognized and extensively studied examples of such complexes polyclonal antibodies against a His6-tag RhlB was received as a in bacteria and higher organisms include ribosomes, replisomes, gift from M. Cashel (National Institutes of Health, Bethesda) and proteasomes (9–11). However, notwithstanding the isola- and used for the detection of RhlB helicase in E. coli cells. The tion of multicomponent RNase E-based complexes from E. coli (1–5, 12), there has been no direct evidence that degradosomes Abbreviation: PNPase, polynucleotide phosphorylase. are present in living cells—rather than being formed in vitro as ¶To whom all materials and reprint requests should be addressed. E-mail: aggregates of individual proteins. The question of whether [email protected]. degradosomes actually exist in vivo in E. coli is especially relevant The publication costs of this article were defrayed in part by page charge payment. This in view of evidence that truncated RNase E protein lacking the article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. C-terminal half is sufficient for cell viability and for RNA §1734 solely to indicate this fact. degradation and processing in vivo in E. coli (13, 14), that RNase Article published online before print: Proc. Natl. Acad. Sci. USA, 10.1073͞pnas.011535498. BIOCHEMISTRY E homologs in certain other bacteria do not contain the scaffold Article and publication date are at www.pnas.org͞cgi͞doi͞10.1073͞pnas.011535498 PNAS ͉ January 2, 2001 ͉ vol. 98 ͉ no. 1 ͉ 63–68 Fig. 1. Quantitation of degradosome proteins in E. coli cell extracts. Coomassie blue stained SDS͞PAGE gels containing different amounts of BSA, as indicated, for quantitation of degradosome protein components (A Upper). By using a known amount of degradosome protein component deduced from the upper panel and anti-PNPase antibody, the amount of endogenous cellular PNPase was detected in log phase (Log), early stationary phase (ES), and late stationary phase (S) by Western blotting (A Lower). (B) Quantitation of individual proteins in mmol͞OD600 of cell culture. Cell lysates were prepared from cells in logarithmic phase at OD600 about 0.5, in early stationary phase at OD600 about 1.0, and in late stationary phase at OD600 about 2.3. Molecular weights (kDa) of individual protein standards are shown. Proteins were quantified by integration of band density area by using LAS-1000 plus (Fuji) and calibration curves, which were constructed from known amounts of BSA and degradosome proteins. M, protein molecular weight standards; DPC, degradosome protein components. specificity of individual antibodies was tested by using cell lysates respectively; 75% and 100% LR-Gold solutions at Ϫ20°C for 4 h from E. coli cells that overexpressed each antigen. and overnight, respectively. The embedded samples were trans- ferred into embedding capsules (PELCO, Redding, CA), poly- SDS͞PAGE, Western Blotting, and Quantitation. Proteins of com- merized in a PELCO UVC1 Cryo chamber at Ϫ20°C for 24 h, plexes in E. coli cell extracts were separated on 8% gels con- and then continuously hardened in the PELCO UVC1 Cryo taining 0.1% SDS. Gels were stained by Coomassie brilliant blue chamber at room temperature for 2–3 d. Embedded samples to visualize protein bands. The ECL Western blot detection were stored in a dehumidifying chamber at room temperature system (Amersham Pharmacia) and LAS-1000 plus (Fuji) were until use.