6.2 Methods
6.2.17 In vitro ribosome-translocon binding analyses
Generation of salt-stripped rough microsomes: Puromycin and high salt-stripped rough microsomes were prepared from temperature-sensitive sterile C. elegans mutants (CB4037). Frozen worms were ground to powder in a mortar and the powder was resuspended in lysis buffer (30 mM HEPES KOH pH 7.4, 75 mM KAc, 5 mM MgCl2, 5 % (w/v) mannitol, 2 mM beta-mercaptoethanol, 1x TmComplete). Samples were sonicated (6x level 2, 50 % duty cycle, Branson sonifier) followed by centrifugation at 20.000 x g for 20 min at 4 °C. Membrane pellets were resuspended in lysis buffer supplemented with 0.008 % (w/v) digitonin (Calbiochem), incubated for 5 min on ice and centrifuged again. Membrane pellets were resuspended in PK-lysis buffer (50 mM HEPES KOH pH 7.4, 1 M KAc, 2,5 mM MgAc2, 1 mM DTT, 1 mM Puromycin, 1x TmComplete) and samples were again sonicated (four times) followed by centrifugation at 20.000 x g for 20 min at 4 °C.
Pelleted membranes were resuspended in PK-lysis buffer, incubated for additional 2 h at 25 °C and pelleted again by centrifugation. Stripped microsomes were washed once in reaction buffer (20 mM NaP pH 7.5, 120 mM NaCl, 6 mM MgCl2, 2 mM DTT, 1x TmComplete), pelleted by centrifugation and resuspended in the same buffer to a final protein concentration of 15 µg/µl.
Binding of ribosomes to microsomes: Stripped microsomes (75 µg protein) were incubated in the presence and absence of C. elegans ribosomes (0.5 µM) and purified wild type or mutant NAC protein (2 µM) for 2 h at 25 °C with constant shaking in a total reaction volume of 35 µl in reaction buffer (20 mM NaP pH 7.5, 120 mM NaCl, 6 mM MgCl2, 2 mM DTT, 1x TmComplete). Subsequently, microsomes were again isolated by centrifugation. The supernatant containing unbound ribosomes was collected and the remaining pellet containing microsome-bound ribosomes was washed once in reaction buffer and then resuspended in 35 µl SDS lysis buffer (62.5 mM Tris pH 6.8, 1 mM EGTA, 2 % (v/v) SDS, 10 % (w/v) sucrose). Equal volumes of both fractions were subjected to SDS-PAGE and immunoblot analysis.
Generation of native rough microsomes: Native rough microsomes were prepared by resuspending worm powder in lysis buffer (30 mM HEPES KOH pH 7.4, 75 mM KAc, 5 mM MgCl2, 5 % (w/v) mannitol, 2 mM beta-mercaptoethanol, 1x TmComplete) followed by sonication on ice (four times) and centrifugation at 20.000 x g for 20 min at 4 °C. Membrane pellets were resuspended in lysis buffer supplemented with 0.008 % (w/v) digitonin, incubated for 5 min on ice and centrifuged again. After one additional washing step in lysis buffer, the native microsome fraction was resuspended in reaction buffer (20 mM NaP pH 7.5, 120 mM NaCl, 6 mM MgCl2, 2 mM DTT, 1x TmComplete) to a final protein concentration of 15 µg/µl.
Detachment of ribosomes from native rough microsomes: Native rough microsomes (75 µg protein) were incubated in 35 $l total reaction volume in the presence or absence of 1 mM puromycin and purified wild type or mutant NAC protein (0.5 µM) for 2 h at 25 °C with constant shaking. Subsequently, microsomes were re-isolated by centrifugation and the ribosome content in the pellet and supernatant fractions was assessed by immunoblotting.
"
CytoQ Cytosolic Quality control compartment
ERAD ER-Associated protein Degradation
eRF eukaryotic RF
Abbreviations
snoRNP small nucleolar RiboNucleoProtein particle
SPase Signal Peptidase
SPC/E Extended Simple Point Charge model SRP Signal Recognition Particle
SUMO Small Ubiquitin related MOdifier TAE Tris Acetate with EDTA
"
$$%"
8 List of figures
Figure 1 - Pathways of the cellular proteostasis network ... 1 Figure 2 - Structure and architecture of the eukaryotic 80S ribosome ... 3 Figure 3 - Composition of bacterial and eukaryotic ribosomes and the common core ... 4 Figure 4 - The ribosomal tunnel and exit site of prokaryotic and eukaryotic ribosomes ... 6 Figure 5 - Co-translational processes on the nascent chain ... 8 Figure 6 - Ribosome assembly of Saccharomyces cerevisiae ... 11 Figure 7 - Simplified scheme of synthesis and processing of eukaryotic rRNA ... 12 Figure 8 - The heat shock response and ER unfolded protein response ... 20 Figure 9 - Classes of molecular chaperones ... 24 Figure 10 - Structure and conformational cycle of Hsp70s ... 27 Figure 11 - Allosteric Hsp70 cycle and cofactor interaction ... 28 Figure 12 - Ribosome-associated factors in different kingdoms of life ... ... 31 Figure 13 - Domain structure of the different subunits of the yeast nascent polypeptide-
associated complex ... 32 Figure 14 - Domain structure of the yeast Ssb-RAC system and mammalian homologues ... 34 Figure 15 - Two positively charged regions that differ between the yeast Hsp70s Ssa
and Ssb might be involved in ribosome binding of Ssb ... 43 Figure 16 - Deletion of 13 C-terminal residues of Ssb influences ribosome binding but
does not hamper growth ... 44 Figure 17 - Stepwise deletion of the C-terminus of Ssb1 or substitution of basic to neutral
charge negatively influences ribosomal interaction ... 45 Figure 18 - Deletion of Ssb1 residues 601-13 abolishes ribosome binding in vitro ... 46 Figure 19 - Deletion of the 13 C-terminal residues affects the structural stability of the
CTD of Ssb ... 47 Figure 20 - A basic region within the SBD of Ssb contributes to its ribosomal interaction ... 48 Figure 21 - In vivo characterization of the ribosome-binding mutant Ssb1#601-13 shows
full complementation of the pleiotropic phenotype of ssb1,2! cells ... 49 Figure 22 - The Ssb1#601-13 ribosome-binding mutant needs its cofactor RAC for
in vivo functionality ... 51 Figure 23 - Fusion constructs containing the C-terminal ribosome-binding site of Ssb1
do not interact with ribosomes in vivo ... 53 Figure 24 - Simulated structures of DnaK and Ssb show qualitatively identical
substrate binding ... 54 Figure 25 - Wild type and mutant Ssb1 interact with canonical Hsp70 substrate
peptides in vitro... 55 Figure 26 - Ssb interacts specifically with the APPY peptide, but binding is less efficient
than the DnaK-APPY interaction ... 56 Figure 27 - Wild type and mutant Ssb1 interact with sigma32 peptide in vitro and show a
DnaK-like peptide release ... 57 Figure 28 - Model of the multilayered interactions of the Hsp70 Ssb at the ribosomal exit site ... 59
List of figures
" $$&"
Figure 29 - Ssb interacts with ssb1,2! aggregates ex vivo and binds ribosomal peptide
substrates in vitro ... 64 Figure 30 - Ssb1 interacts with r-peptides that are similar to canonical Hsp70 substrates
but disfavors acidic model peptides ... 65 Figure 31 - Ssb deletion blocks efficient 35S rRNA processing in the nucleolus ... 66 Figure 32 - Ssb1,2! cells shows phenotypes similar to yeast strains lacking typical
ribosome biogenesis factors ... 67 Figure 33 - Ssb interacts with factors involved in ribosomal function, architecture
and synthesis ... 69 Figure 34 - The interaction of Ssb1-TAP with ribosomal and other proteins is ATP-dependent ... 70 Figure 35 - Ssb can be detected at several ribosomal precursor complexes in the nucleolus,
the nucleoplasm and the cytosol ... 72 Figure 36 - Selected interactions of Ssb and other factors with ribosomal assembly intermediates ... 74 Figure 37 - Ssb contains predicted nuclear localization signals, a functional but non-essential
nuclear export sequence and partially localizes to the nucleus ... 75 Figure 38 - Ssb1 fused to a nuclear localization signal (NLS) is depleted from the cytosol and
partially complements growth ... 77 Figure 39 - Nuclear localized Ssb is functional during ribogenesis and synthesis of r-proteins
but displays deficits in translation ... 79 Figure 40 - Purified wt NAC but not the RRK-AAA mutant binds to C. elegans ribosomes ... 87 Figure 41 - Ribosome-bound NAC prevents interaction of the translation machinery with
microsomes ... 88 Figure 42 - Ribosome-bound NAC enhances puromycin induced detachment of ribosomes
from microsomes ... 89 Figure 43 - A double secure mechanism sustains ER targeting specificity in vivo ... 90