EMBO/soda-nel huggingface dataset:
Optimal Sidestepping of Intraflagellar Transport Kinesins Regulates Structure and Function of Sensory Cilia (2020)
Figure 2

Fig. 2. Sidesteps of anterograde IFT-particles

A. Frame series shows the off-axis movement of an IFT-particle (#1). A neighboring particle (#2) serves as a fiduciary marker. Scale bar: 200 nm.

B. Representative two-dimensional IFT-tracks from WT, kinesin-II (klp-11), and osm-3 null animals. Red and violet circles indicate the start and end of the tracks, respectively. Proteins used for tracking experiments are CHE-3 tagged with 7x GFP, or DYF-11 tagged with 3x GFP.

C. Time-averaged mean-square displacement (MSD) versus lag time (tlag) of 10-15 tracks from WT and IFT-kinesin mutant animals. Proteins used for tracking experiments are CHE-3 tagged with 7x GFP, CHE-3 tagged with 7x GFP plus DYF-11 tagged with 3x GFP, CHE-2 tagged with 7x GFP, or DYF-11 tagged with 3x GFP. Error bars indicate standard errors.

D. Angle histogram (rose plot) of the deviation angles of displacement in 10-15 tracks from WT and mutant animals. The deviation angle is defined as the angle between the displacement vector and the overall direction of motion. Deviation to the right or left of the overall direction is between 0° and 180° or between 0° and -180°, respectively. Deviations between 90° ~ 180° and -90° ~ -180° are backstepping (orange). Proteins used for tracking experiments are the same as in Fig. 1E.

E. Average deviation angles in 10-15 tracks from WT and mutant animals. Comparisons were performed with the WT. Proteins used for tracking experiments are the same as in Fig. 1E. *p<0.05, **p<0.01, ****p < 0.0001 by Student's t-tests. Error bars indicate standard deviations.

F. Representative tracks of the recombinant C. elegans kinesin-II and OSM-3-kinesin moving along suspended MTs in the optical trap assay. Appendix Figures S3E depicts the experiment scheme.

G. Representative TEM images of the middle ciliary segments from WT and IFT-kinesin mutant animals. Red arrowheads indicate ectopic singlet microtubules. Blue arrowheads indicate ectopic doublet microtubules. The schematics below depict the phenotype of each image. Scale bars: 100 nm.
Optimal Sidestepping of Intraflagellar Transport Kinesins Regulates Structure and Function of Sensory Cilia (2020)
Figure 3

Fig. 3. GS insertion of OSM-3 increased sidestepping

A. Design of OSM-3 constructs with flexible necks. Top, poly-GS repeats (orange) were knocked into the endogenous osm-3 locus. Bottom, schematic of osm-3 gene model, and sgRNA sequence.

B. Representative two-dimensional IFT tracks from WT and OSM-3(1 or 3xGS) animals. Red and violet circles indicate the start and end of the tracks, respectively. Proteins used for tracking experiments are the same as in Fig. 1E.

C. Time-averaged mean-square displacement (MSD) versus lag time (tlag) of 10-15 tracks from WT and mutant worms. Proteins used for tracking experiments are the same as in Fig. 1E. Error bars indicate standard errors.
Optimal Sidestepping of Intraflagellar Transport Kinesins Regulates Structure and Function of Sensory Cilia (2020)
Figure 4

Fig. 4. GS insertion of OSM-3 reduced IFT speeds and perturbed ciliary ultrastructure

A. Histogram of anterograde IFT speeds of 3xGFP::CHE-3 at the middle (top) and distal (bottom) ciliary segments (top). The plots were fit by a Gaussian distribution. Comparisons were performed between the WT and osm-3 mutants. ***p < 0.001 by Student's t-tests. Error bars indicate standard errors.

B. Schematic depiction of the C. elegans amphid and phasmid cilia. Each cilium contains a ciliary base, a middle segment (m.s.), and a distal segment (d.s.).

C. Amphid (top) and phasmid (bottom) cilia in the WT and osm-3 GS knock-in animals. Cilia are marked with 7xGFP::CHE-3. Arrowheads indicate the ciliary base. Arrows indicate the junctions between the middle and distal segments. Scale bars: 5 μm.

D. Cilium length (mean ± SD) in WT and osm-3 mutant animals. Comparisons were performed between the WT and osm-3 mutants. N = 24-32. n.s., not significant by Student's t-tests. Error bars indicate standard errors.

E. Quantifications of dye-filling defects from WT and osm-3 mutant animals. N ≥ 100.

F. Representative TEM images of the middle ciliary segments from osm-3(1xGS) and osm-3(3xGS) worms. Red arrowheads indicate ectopic singlet microtubules. Blue arrowheads indicate ectopic doublet microtubules. The schematics below depict the phenotype of each image. Scale bars: 100 nm.
Dopamine signaling promotes the xenobiotic stress response and protein homeostasis (2016)
Figure 1

Figure 1. Dopaminergic signaling modulates protein turnover

A Schematic representation of the UbG76V-GFP chimeric repoter and its associated internal control (mRFP or mCherry, in red). The amino acid sequences for ubiquitin are in yellow, whereas the sequences for GFP are in green. The UbG76V-GFP chimera, which contains a mutation in the terminal residue of ubiquitin, cannot be cleaved. The resulting protein is a substrate for poly-ubiquitination (indicated by the circles labeled with “u”) and degradation by the proteasome. High levels of reporter poly-ubiquitination and/or proteasome activity result in little or no GFP fluorescence.

B Quantified fluorescence of UbG76V-GFP normalized to mRFP in the hypodermis from animals of the indicated timepoint (in hours) after the L4 stage. Animals have been exposed to the indicated knockdown RNAi bacterial strains or a strain that only contains the empty RNAi vector. *P<0.001, Student t-test corrected for multiple comparisons at each time point using the Holm-Sidek method. N=20 animals per genotype and timepoint. Error bars indicate SEM.

C,D Co-expression via the col-19 promoter of UbG76V-GFP (green) and mRFP (red) in C. elegans hypodermis from a single integrated transgene at L4+48 hours. Wild-type animals are shown. Bar, 50 microns. UbG76V-GFP fluorescence is barely detectable.

E,F Co-expression via the sur-5 promoter of UbG76V-GFP (green) and mCherry (red) in C. elegans from two separate integrated transgenes (with focus on the intestine) at L4+48 hours. Wild-type animals are shown. Bar, 50 microns. UbG76V-GFP fluorescence is barely detectable.

G,H Co-expression of UbG76V-GFP (green) and mRFP (red) in C. elegans hypodermis at L4+48 hours in dop-1(vs100) mutants. Bar, 50 microns. Abundant, stable UbG76V-GFP fluorescence is observed.

I,J Co-expression of UbG76V-GFP (green) and mCherry (red) in C. elegans from the sur-5 promoter at L4+48 hours in dop-1(vs100) mutants. Bar, 50 microns. Abundant, stable UbG76V-GFP fluorescence is observed, particularly in the intestine.

K Quantified fluorescence of UbG76V-GFP normalized to mRFP in the hypodermis from animals of the indicated timepoint (in hours) after the L4 stage and of the indicated genotype. *P<0.001, Student t-test corrected for multiple comparisons at each time point using the Holm-Sidek method. N=20 animals per genotype and timepoint. Error bars indicate SEM.

L Quantified fluorescence of UbG76V-GFP normalized to mRFP in the hypodermis of animals at L4+48 hours and of then indicated genotypes. ***P<0.001, ANOVA with Dunnetts posthoc comparison to the wild-type control. N=20 animals per genotype and timepoint. Error bars indicate SEM.

M Quantified fluorescence of UbG76V-GFP normalized to mCherry (both expressed by the sur-5 promoter) in the intestine of animals at L4+48 hours and of then indicated genotypes. ***P<0.001, **P<0.01, *P<0.05, ANOVA with Dunnetts posthoc comparison to the wild-type control. N=20 animals per genotype and timepoint. Error bars indicate SEM.
Dopamine signaling promotes the xenobiotic stress response and protein homeostasis (2016)
Figure 2

Figure 2. Dopaminergic signaling promotes protein poly-ubiquitination

A Western blot of lysed nematodes at the L4+48 hour stage, probed with antibodies recognizing, ubiquitin, GFP, RPN-11, multiple 20S proteasome subunits, or actin as a loading control. Animals express UbG76V-GFP protein in their hypodermis. The position of molecular weight markers is shown on the left of each blot. The position of UbG76V-GFP protein, as well as UbG76V-GFP with the indicated number of additional ubiquitin moieties, based on molecular weight, is indicated on the right of each blot. Twenty animals were loaded per lane for each indicated genotype (WT indicates wild type).

B Western blot of lysed nematodes at the L4+48 hour stage, probed and annotated as in (A). Animals express UbG76V-GFP protein from the sur-5 promoter. Thirty animals were loaded per lane for each indicated genotype (WT indicates wild type).

C Quantified fluorescence of UbG76V-GFP normalized to mRFP from lysates of transgenic nematodes at L4+48 hours that express these reporters in the hypodermis. Animals were exposed to bacteria containing the indicated RNAi clone or an empty RNAi vector as a control. *P<0.05, ANOVA with Dunnetts posthoc comparison to the empty RNAi vector control. N=3 trials. Error bars indicate SEM.

D Quantified epoxomicin-sensitive proteasome activity (as measured through the turnover of fluorescent chymotrypsin substrate) from the same lysates and exposed to the same RNAi as in (C). *P<0.05, ANOVA with Dunnetts posthoc comparison to the empty RNAi vector control. N=3 trials. Error bars indicate SEM.

E Quantified fluorescence of UbG76V-GFP normalized to mRFP from lysates of transgenic nematodes at L4+48 hours that express these reporters in the hypodermis. Animals were of the indicated genotype. ***P<0.001, **P<0.01, *P<0.05, ANOVA with Dunnetts posthoc comparison to wild type. N=4 trials. Error bars indicate SEM.

F Quantified epoxomicin-sensitive proteasome activity (as measured through the turnover of fluorescent chymotrypsin substrate) from the same lysates as in (E). No statistical difference (P<0.05 cutoff) was detected by ANOVA. N=4 trials.

G Percentage of live animals of the indicated genotype assayed after 6-hour exposure to heat shock. 100% of control animals of the same genotype but not given heat shock survived. *P<0.01, Student t-test. N=3 trials. Error bars indicate SEM.

H Percent of animals of the indicated genotype containing aggregates of Poly(Q)44::YFP at the indicated time (in days) after L4 stage. *P<0.01, Student t-test. N=3 trials, 50-80 animals per trial per day. Error bars indicate SEM.
Dopamine signaling promotes the xenobiotic stress response and protein homeostasis (2016)
Figure 3

Figure 3. Dopamine signaling promotes protein turnover via the xenobiotic stress pathway

A Relative abundance of mRNA for the indicated xenobiotic stress response gene (or UbG76V-GFP or mRFP) detected in wild type versus dop-1 mutants. Values are based on RNA-seq/EdgeR analysis, with the relative levels for each gene normalized to the value observed in the wild-type control. ****P<0.0001, **P<0.01, *P<0.05 FDR from EdgeR analysis using a Benjamini and Hochberg correction.

B,C Quantified fluorescence of UbG76V-GFP normalized to (B) mRFP in the hypodermis or (C) mCherry in the intestine from L4+48 hour animals exposed to feeding RNAi for either an empty vector control or the indicated xenobiotic stress gene. ****P<0.0001, ***P<0.001, **P<0.01, ANOVA, Tukey’s multiple comparison test compared to control. N=20 animals per genotype and timepoint. Error bars indicate SEM.

D Percentage of live animals raised on the indicated feeding RNAi (or empty vector control) bacteria assayed after 6-hour exposure to heat shock. 100% of control animals of the same genotype but not given heat shock survived. ***P<0.001, **P<0.01, *P<0.05, ANOVA, Tukey’s multiple comparison test compared to wild type. N=3 trials. Error bars indicate SEM.

E Quantified fluorescence of UbG76V-GFP normalized to mRFP in the hypodermis from L4+48 hour animals of the indicated genotype. ****P<0.0001, ANOVA, Tukey’s multiple comparison test compared to wild type. N=20 animals per genotype and timepoint. Error bars indicate SEM.

F Percentage of live animals of the indicated genotype assayed after 6-hour exposure to heat shock. 100% of control animals of the same genotype but not given heat shock survived. **P<0.01, *P<0.05, ANOVA, Tukey’s multiple comparison test compared to wild type. N=3 trials. Error bars indicate SEM.

G Percent of animals of the indicated genotype containing aggregates of Poly(Q)44::YFP at the indicated time (in days) after L4 stage. *P<0.001, Student t-test corrected for multiple comparisons at each time point using the Holm-Sidek method. N=3 trials, 50-80 animals per trial per day. Error bars indicate SEM.
Dopamine signaling promotes the xenobiotic stress response and protein homeostasis (2016)
Figure 4

Figure 4. Dopamine signaling modulates xenobiotic stress survival

A Quantified fluorescence of UbG76V-GFP normalized to mRFP in the hypodermis from L4+48 hour animals of the indicated genotype. ****P<0.0001, ANOVA, Tukey’s multiple comparison test compared to wild type. N=20 animals per genotype. Error bars indicate SEM.

B Quantified fluorescence of UbG76V-GFP normalized to mRFP in the hypodermis from either L4+24 hour animals (left half of graph) or L4+48 hour animals (right half of graph) of the indicated genotype. Animals were either continually grown on regular OP50 food (gray bars) or switched to PA14 food at the L4 stage (orange bars). ****P<0.0001, ***P<0.001, **P<0.01, ANOVA, Tukey’s multiple comparison test compared to wild type. N=20 animals per genotype. Error bars indicate SEM.

C Survival curves (percentage of live animals assayed each hour after PA14 exposure) for the indicated mutants at 20oC. Plog-rank<0.0001. Median survival time was 90 hours for wildtype (N=304), 72 hours for dop-1(vs100) (N=180), 78 hours for elt-3(gk121) (N=120), 78 hours for pqm-1(ok485) (N=116), 78 hours for nhr-28(gk568) (N=123), and 42 hours for skn-1(tm3411) (N=33). Eight wild-type animals and six dop-1 mutants were censored (the animals crawled up the side of the plate and dessicated). The media contained 50 µg/ml of 5-fluoro-2′-deoxyuridine (FUdR to prevent the growth of egg offspring during the experiment.

D Quantified fluorescence of UbG76V-GFP normalized to mRFP in the hypodermis from either L4+48 hour animals (left half of graph) or L4+72 hour animals (right half of graph) of the indicated genotype. Animals were either continually grown on regular OP50 food (gray bars) or switched to NGM plates containing 1 mM paraquat at the L4 stage (orange bars). ****P<0.0001, ***P<0.001, ANOVA, Tukey’s multiple comparison test compared to wild type. #P<0.001 ANOVA, Bonferoni multicomparison as indicated by the bracketed lines. N=20 animals per genotype. Error bars indicate SEM. No UbG76V-GFP fluorescence was detected in wild type at L4+72 hours on either OP50 or switched to NGM plates containing 1mM paraquat.
Diagnostic host gene signature for distinguishing enteric fever from other febrile diseases (2019)
Figure 5

Figure 5: Prediction of Nepali unknown samples using the 2-class and qPCR validation. (a) PCA of sEF samples based on the 5-gene signature (based on gene array data) coloured by predicted class membership (EF: purple; green: rest). (b) Dot plot of prediction probability of being class EF versus the expression score calculated on the bases of the 5-gene signature (based on gene array data). (c) qPCR gene expression scores of the 5-gene signature (∆∆CT over PPIA) for CTRLs, 03NP-sEF, 03NP-SPT and 03NP-ST samples from Nepal. Yellow diamonds in the 03NP-sEF category represent the 9 patients classified as EF based on the random forest algorithm. (d) qPCR expression values (∆∆Ct over PPIA) of the 5-gene signature in control samples (Oxford and Nepal), S. Paratyphi A (03NP-SPT) or S. Typhi (03NP-ST) in Nepal, samples at day 7 after challenge of participants who stayed well following challenge with S. Typhi (nD7), or typhoid diagnosis after challenge (TD) in the Vi-TCV study (Table S2). Colour legend in panel (e). (e) Combined qPCR expression score of the 5-gene signature. Black arrows indicate outlier samples. (f) Temperature and CRP for samples of which data was available (CRP was only measured in the Oxford CHIM). D0 = pre-challenge baseline Vi-TCV study; nD7 = day 7 samples of participants who stayed well following challenge (Vi-TCV study); SPT = S. Paratyphi A (03NP); ST = S. Typhi (03NP); TD = Typhoid diagnosis (Vi-TCV study). (g) Spearman's rank correlation of the 5-gene combined expression score and (left) temperature (only nD7 and TD samples from the Oxford CHIM - Vi-TCV and SPT and ST cases from Nepal at presentation to hospital were included) and (right) CRP (CRP was only available for Oxford CHIM - Vi-TCV samples and we excluded D0 baseline measures).
A SNARE geranylgeranyltransferase essential for the organization of the Golgi apparatus (2020)
Figure 1

Figure 1. PTAR1 exists as a heterodimeric complex with the β Subunit of RabGGTase.

A PTAR1 protein expression in rat tissues. Proteins extracted from various rat tissues (15 μg each) were analyzed by immunoblotting with antibodies against PTAR1.

B Identification of RabGGTβ as a binding protein of PTAR1. Anti-FLAG immunoprecipitates from control HeLa S3 cells or FLAG-PTAR1-expressing HeLa S3 cells were analyzed by SDS-PAGE and silver staining. The 35 kDa protein was identified as RabGGTβ by mass spectrometry. The asterisk denotes a degradation product of FLAG-PTAR1.

C Co-immunoprecipitation of endogenous PTAR1 and RabGGTβ from rat brain and thymus cytosol.

D Fractionation of rat brain cytosol by Mono Q anion exchange column chromatography. Column fractions were analyzed by immunoblotting with antibodies against RabGGTα, PTAR1, and RabGGTβ. RabGGTβ eluted in two peaks (peak A and peak B).

E Co-immunodepletion of PTAR1 and RabGGTβ by anti-PTAR1 IgG. PTAR1 was immunodepleted from the Mono Q peak A fraction using anti-PTAR1 IgG-bound protein A beads, and the supernatant was analyzed by immunoblotting with antibodies against PTAR1 and RabGGTβ. Normal rabbit IgG and anti-PTAR1 IgG preabsorbed with the antigen peptide were used as controls.

F Fractionation of the Mono Q peak A fraction by Superdex 200 gel filtration chromatography. Column fractions were analyzed by immunoblotting with antibodies against PTAR1 and RabGGTβ.

G Gel filtration chromatography of the recombinant PTAR1-RabGGTβ complex purified from Sf9 insect cells on a Superdex 200 column. Inset shows SDS-PAGE and Coomassie staining analysis of the purified complex.

H Schematic diagram of the protein prenyltransferase family. FTase and GGTase-I share the same α subunit. RabGGTase and the PTAR1-RabGGTβ complex (GGTase-III) share the same β subunit.
A SNARE geranylgeranyltransferase essential for the organization of the Golgi apparatus (2020)
Figure 2

Figure 2. Identification of Ykt6 as a substrate of GGTase-III.

A Molecular structures of geranylgeranyl pyrophosphate (GGPP) and its biotinylated analogue biotin-geranyl pyrophosphate (BGPP). Geranylgeranyl moiety consists of four repeats of 5-carbon isoprene unit.

B Purification strategy for GGTase-III substrate proteins using statin and BGPP. "X" represents a putative substrate protein of GGTase-III. HMG-CoA, hydroxymethylglutaryl-CoA.

C Identification of Ykt6 as a protein biotin-geranylated by GGTase-III. Cytosolic proteins extracted from statin-treated HeLa S3 cells were applied to GST or GST-GGTase-III affinity columns. Bound proteins were eluted and incubated with recombinant GGTase-III and BGPP. Reaction products were separated by SDS-PAGE, transferred to a nitrocellulose membrane, and probed with horseradish peroxidase (HRP)-labeled avidin to detect biotinylated proteins. Mass spectrometry identified the 25 kDa protein as Ykt6.

D Biotin-geranylation of recombinant Ykt6 by GGTase-III. Bacterially produced recombinant untagged Ykt6 (left) was incubated with buffer, GGTase-III, or RabGGTase in the absence or presence of BGPP for 30 min at 37ºC. Biotin-geranylated Ykt6 was detected as in (C).
A SNARE geranylgeranyltransferase essential for the organization of the Golgi apparatus (2020)
Figure 3

Figure 3. GGTase-III geranylgeranylates Cys194 of Cys195-farnesyl Ykt6.

A Schematic diagram showing the domain structure of human Ykt6 protein. Ykt6 has two cysteine residues, Cys194 and Cys195, at the fifth and fourth positions from the C-terminus.

B Alignment of the C-terminal amino acid sequences of Ykt6 from various species. The conserved tandem cysteines are highlighted in gray.

C Farnesylation of recombinant Ykt6 by FTase. Recombinant Ykt6 (5 μM) was incubated with FTase (0 or 50 nM) and 3H-FPP (1 μM; ~3,500 dpm/pmol) at 37ºC. Reactions were stopped at the indicated time points, and the amount of 3H-farnesyl transferred to Ykt6 was quantified by scintillation counting (mean ± SEM, n = 3).

D Geranylgeranylation of recombinant Ykt6 by GGTase-III. Recombinant Ykt6 (5 μM) was incubated with increasing concentrations of GGTase-III (0-100 nM) and 3H-GGPP (1 μM; ~3,000 dpm/pmol) for the indicated times, and the amount of 3H-geranylgeranyl transferred to Ykt6 was quantified (mean ± SEM, n = 3).

E Geranylgeranylation of Cys to Ser mutants of Ykt6 by GGTase-III. WT and mutant Ykt6 proteins (5 μM each) were incubated with GGTase-III (100 nM) and 3H-GGPP (1 μM) for the indicated times, and the amount of 3H-geranylgeranyl transferred to Ykt6 was quantified (mean ± SEM, n = 3).

F Sequential prenylation process of Ykt6. SAM, S-adenosylmethionine; SAH, S-adenosylhomocysteine; PPi, inorganic pyrophosphate.

G Mass spectra of prenylated Ykt6 peptides. Recombinant Ykt6 was farnesylated by FTase (FTase), further processed by RCE1 and ICMT (RCE1/ICMT), and finally geranylgeranylated by GGTase-III (GGTase-III). After purification to homogeneity, the prenylated Ykt6 proteins were digested with chymotrypsin (left) or V8 protease (right) and analyzed by MALDI-TOF mass spectrometry. The calculated m/z values for the C-terminal peptide ions are shown in parentheses. The peaks labeled with an asterisk correspond to amino acids 14-38 of Ykt6 (AKVVLLKAAYDVSSFSFFQRSSVQE; m/z 2806.48).

H Effects of C-terminal processing and Cys195 farnesylation on Cys194 geranylgeranylation by GGTase-III. Purified recombinant unprenyl Ykt6, Cys195-farnesyl Ykt6, and Cys195-farnesyl Ykt6ΔAIM (5 μM each) were incubated with GGTase-III (100 nM) and 3H-GGPP (1 μM) for the indicated times, and the amount of 3H-geranylgeranyl transferred to Ykt6 was quantified. (mean ± SEM, n = 3).

I Gel filtration chromatography of purified unprenyl Ykt6, Cys195-farnesyl Ykt6ΔAIM, and Cys194/195-diprenyl Ykt6ΔAIM (135 μg each) on a Superdex 200 column. Dotted lines indicate the elution profile of unprenyl Ykt6 for comparison. The prenylated Ykt6 proteins were eluted slightly later than the unprenylated Ykt6, suggesting more compact and stable folds.
A SNARE geranylgeranyltransferase essential for the organization of the Golgi apparatus (2020)
Figure 4

Figure 4. Crystal structure of apo-GGTase-III and its complex with Cys195-farnesyl Ykt6ΔAIM and GGPP.

A Structural comparison of GGTase-III and other prenyltransferases. The α and β subunits of the prenyltransferases are shown in green and cyan, respectively. The unique N-terminal domain of PTAR1 is shown in yellow.

B Overall structure of GGTase-III complexed with Cys195-farnesyl Ykt6ΔAIM and GGPP in two orientations. GGTase-III is colored as in (A). The longin domain of Ykt6 is in pink and SNARE domain is in magenta.

C, D Magnified views of site 1 (C) and site 2 (D) depicting the interaction between PTAR1 and Ykt6. Dashed lines indicate hydrogen bonds.

E Geranylgeranylation activity of WT GGTase-III and the indicated mutants (100 nM each). Cys195-farnesyl Ykt6ΔAIM (1 μM) and 3H-GGPP (1 μM) were used as substrates (mean ± SEM, n = 3).

F Kinetic analysis of WT GGTase-III and the site 1 or site 2 mutant (100 nM each) using increasing concentrations of Cys195-farnesyl Ykt6ΔAIM (mean ± SEM, n = 3 for WT and Y306A, n = 1 for V48A/V50A).

G Geranylgeranylation of unprenyl WT Ykt6 and the site 1 or site 2 mutants by GGTase-III. WT and mutant Ykt6 proteins (5 μM each) were incubated with GGTase-III (100 nM) and 3H-GGPP (1 μM), and the amount of 3H-geranylgeranyl transferred to Ykt6 was quantified (mean ± SEM, n = 3).