QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online 13 May 2008
doi: 10.1242/jcs.020321

This Article Summary Full Text Full Text (PDF) Supplementary Material Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Favaloro, V. Articles by Dobberstein, B. Search for Related Content PubMed PubMed Citation Articles by Favaloro, V. Articles by Dobberstein, B. Social Bookmarking                         What's this?

Distinct targeting pathways for the membrane insertion of tail-anchored (TA) proteins

Vincenzo Favaloro^*, Milan Spasic^*,, Blanche Schwappach and Bernhard Dobberstein^¶

Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH), DKFZ-ZMBH Allianz, Im Neuenheimer Feld 282, D-69120 Heidelberg, Germany

View larger version (41K): [in this window] [in a new window]   Fig. 1. Post-translational membrane insertion of R4op. (A) Topology of RAMP4 in the ER membrane. RAMP4 is a tail-anchored ER membrane protein that exposes its N-terminus on the cytosolic and the C-terminus on the lumenal side of the membrane. (B) Schematic representation of RAMP4 and RAMP4op (R4op). R4op contains at its C-terminus a bovine opsin tag comprising 13 amino acid residues (dark grey box). The tag provides an N-glycosylation site (fork). The predicted transmembrane domain (TMD) is represented as a black box. A single cysteine residue in the TMD is typed in bold. (C and D) In vitro translation and membrane insertion of R4op and the type II membrane protein Invariant chain (Ii), respectively. Proteins were synthesised in rabbit reticulocyte lysate (RRL), in the absence (lanes 1, 4, 5 and 6) or presence (lanes 2 and 3) of rough microsomes (RM co). Rough microsomes were added after completion of translation (RM post) to samples shown in lanes 4 and 5. Where indicated, samples were treated with EndoH to remove N-linked oligosaccharides. Proteins were immunoprecipitated using either anti-opsin (C) or anti-Ii (D) antibodies, were separated by SDS-PAGE and visualised by autoradiography. g-R4op: glycosylated R4op; Ii*: non-glycosylated Ii.

View larger version (37K): [in this window] [in a new window]   Fig. 2. Insertion competence and size of the cytosolic R4op complex. (A) Time dependence of R4op insertion into RMs. R4op was synthesised in the RRL, translation was stopped by the addition of puromycin and ribosomes were removed by sedimentation. Reactions were further incubated for the times indicated. RMs were then added and incubation continued for 30 minutes. R4op was immunoprecipitated and characterised by SDS-PAGE and autoradiography. The amounts of immunoprecipitated non-glycosylated R4op (black bars) and glycosylated g-R4op (grey bars) were quantified (see histogram). (B) Sucrose-density-gradient analysis of cytosolic R4op. R4op was synthesised in the RRL. Aliquots were loaded on top of 10-20% sucrose density gradients containing 2 mM ATP (left) or ADP (right). After centrifugation and fractionation, proteins were analyzed by SDS-PAGE and autoradiography. Black arrowheads and numbers above the gel indicate migration positions of proteins used as molecular markers and their molecular mass in kDa, respectively.

View larger version (51K): [in this window] [in a new window]   Fig. 3. Crosslinking of R4op. (A) Crosslinking of cytosolic R4op in the presence or absence of nucleotides. After termination of R4op translation in RRL, nucleotides were removed and either no nucleotides or 2 mM ATP or ADP added. After the crosslinking by either DSS (D) or BMH (B), R4op was immunoprecipitated and characterised by SDS-PAGE and autoradiography. (B) Crosslinking of R4op in the presence or absence of ribosomes. After translation, ribosomes were removed by ultracentrifugation. A total fraction (total), the resuspended pellet (pellet) and the supernatants (Sn) were crosslinked by BMH or left uncrossliked (–). (C) Crosslinking of R4op in the presence of Triton X-100. After synthesis of R4op in the RRL, the samples were adjusted to the indicated amount of Triton X-100 and then BMH was added. (D) Crosslinking of R4op in the absence or presence of RMs. After synthesis of R4op, BMH was added either immediately or after the additional incubation with RMs. g-R4op, glycosylated R4op. R4op x p40, crosslinked product of R4op and a 40-kDa protein.

View larger version (17K): [in this window] [in a new window]   Fig. 4. Membrane requirements for R4op and Ii insertion into PKRM. R4op and Ii were synthesised in the RRL. Membranes washed in high-salt buffer supplemented with puromycin (PKRM), mock-treated (PKRM mock) or treated with 1 or 2 µg/ml trypsin (PKRM-T 1 and PKRM-T 2, respectively) were present during the synthesis of Ii. R4op was incubated with these membranes post-translationally. Where indicated, 100 nM of soluble SRP receptor (SR) was added. Proteins were analysed by SDS-PAGE and autoradiography. Ii*, nonglycosylated Ii; g-R4op, glycosylated R4op.

View larger version (24K): [in this window] [in a new window]   Fig. 5. Identification of p40. (A) Immunoaffinity purification of p40 associated with R4op. Large-volume RRL translation reactions were incubated with R4op mRNA (lanes 1 and 2) or without (lanes 3 and 4). R4op-containing complexes were affinity-purified using anti-opsin antibody beads and proteins released from R4op by elution with 0.1% Triton X-100 (TX) (lanes 1 and 3). Remaining bound proteins were eluted from the column by using an acidic glycine buffer (gly) (lanes 2 and 4). Eluted proteins were separated by SDS-PAGE and silver stained. The protein band of about 40 kDa was cut out, proteins were eluted and peptide sequences determined by mass spectroscopy. Peptide sequences identified p40 as Asna1. (B) Immunoprecipitation of R4op x p40 crosslinked product. R4op was synthesised in RRL and aliquots of the reaction were either crosslinked with BMH (+) or incubated with DMSO solvent alone (–). Aliquots of both reactions were either directly applied to the gel (lanes 1 and 2) or immunoprecipitated by anti-opsin antibody (lanes 3 and 4), an anti-Asna1 antibody (lanes 5 and 6) or a pre-immune serum (lanes 7 and 8) and characterised by SDS-PAGE and autoradiography.

View larger version (56K): [in this window] [in a new window]   Fig. 6. Cytosolic R4op (RAMP4op) and S61βop (Sec61βop) but not b5op (cytochrome-b5op) or Ii (invariant chain) can be crosslinked to Asna1. (A) Outline of the sequences around the TM (black) of R4op, S61βop, b5op and Ii. The sequences are aligned by the relative position of their TM domains. Lysine (K) and cysteine (C) residues that can function in crosslinking with DSS and BMH, respectively, are indicated. (B-E) Crosslinking of the TA proteins and Ii in the RRL: R4op (A), S61βop (B), b5op (C) and Ii (D) were in vitro synthesised in the RRL and small molecules were removed by gel filtration and either DMSO (–) or the crosslinker BMH (B) or DSS (D) were added. The TA proteins were immunoprecipitated with anti-opsin (-op), anti-Asna1 (-Asna-1) or the unrelated anti-Myc (-Myc) antibodies and characterised by SDS-PAGE and autoradiography. R4op x Asna-1 and S61βop x Asna-1, R4op and S61βop, respectively, crosslinked to Asna1. , yet-unidentified complexes of higher molecular mass.

View larger version (62K): [in this window] [in a new window]   Fig. 7. Comparison of the requirements for membrane insertion of R4op, S61βop and b5op. (A) Nucleotide- and redox-state dependence of the post-translational membrane insertion. After the synthesis of the TA proteins in the RRL (lane 1) RMs were added either directly (lane 2) or after treatments as indicated (lanes 3-10). To test nucleotide tri-phosphate (NTP) and redox conditions required for membrane insertion of these TA proteins, small molecules were removed from the lysates by gel filtration (lanes 3-10) and addition of 2 mM H2O2, 2 mM DTT, 3 mM ATP (A), GTP (G) or CTP (C) as indicated. After incubation, proteins were separated by SDS-PAGE and visualised by autoradiography. Glycosylated TA protein (glyc) was quantified in percent (right panel). (B) Nucleotide depletion by apyrase and membrane insertion of R4op, S61βop and b5op. The three TA proteins were synthesised in the RRL (lane 1) and RMs added either directly (lane 2) or after removal of small molecules by gel filtration and addition of H2O2, DTT, apyrase or ATP as indicated (lanes 3-8). (C) Release of R4op from Asna1 and membrane insertion. After synthesis of R4op in the RRL, small molecules were removed by gel filtration and lysates were adjusted to either 2 mM H2O2 (lanes 1-8) or 2 mM DTT (lanes 9-16) and 3 mM ATP and then incubated with RMs as indicated. After the membrane insertion small molecules were removed by gel filtration and BMH crosslinking induced where indicated. Proteins were immunoprecipitated using anti-opsin antibodies (-op), separated by SDS-PAGE and visualised by autoradiography. (D) Free sulfhydryl (SH) groups on cytosolic proteins are required for the membrane insertion of R4op and S61βop but not of b5op. After synthesis of the three TA proteins in the RRL (lane 1) RMs were added (lane 2) and the insertion reaction was incubated for 30 minutes at 30°C. To test whether free SH-groups are needed for membrane insertion, lysates were adjusted to 5 mM NEM where indicated. After incubation, small molecules were removed by gel filtration and the lysates adjusted to 2 mM H2O2 (lanes 3-6), 2 mM DTT (lanes 7-10) and 3 mM ATP as indicated and incubated with RMs. Proteins were separated by SDS-PAGE and visualised by autoradiography.

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

Twitter