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First published online 24 February 2009
doi: 10.1242/jcs.041764

Journal of Cell Science 122, 867-874 (2009)
Published by The Company of Biologists 2009
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Identification of a palmitoyl acyltransferase required for protein sorting to the flagellar membrane

Brian T. Emmer1, Christina Souther1, Krista M. Toriello1, Cheryl L. Olson1, Conrad L. Epting2 and David M. Engman1,*

1 Departments of Pathology and Microbiology-Immunology, Northwestern University Feinberg School of Medicine, Chicago, IL 60611, USA
2 Department of Pediatrics, Northwestern University Feinberg School of Medicine, Chicago, IL 60611, USA


Figure 1
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  		Fig. 1. T. brucei calflagins are palmitoylated in vivo. Palmitoylated proteins were specifically labeled in T. brucei lysates by cleaving fatty acid-thioester bonds with hydroxylamine and labeling the liberated thiols with biotin-HPDP, as described in the Materials and Methods. Acyl-biotin exchange samples from control (Tris) and hydroxylamine (NH2OH) treatment conditions were then subjected to streptavidin affinity chromatography to purify palmitoylated proteins. Samples from each input (I) and eluate (E) fraction were analyzed by immunoblotting. A control palmitoylated protein (CAP5.5) was purified in a hydroxylamine-dependent manner, whereas negative controls from the cytoskeleton (β-tubulin), cytoplasm (Hsp70), endoplasmic reticulum (BiP) and cell membrane (procyclin) were not. The calflagin antiserum identified three bands corresponding to Tb44, Tb17 and Tb24 (indicated) as well as a cross-reactive protein (asterisk) of 38 kDa in the streptavidin input. Only the calflagin proteins were purified by streptavidin under hydroxylamine treatment.

 

Figure 2
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  		Fig. 2. N-terminal mutagenesis abolishes acylation. (A) The N-terminal sequence is highly conserved among the calflagins and is very similar to that of the dually acylated flagellar Ca2+-binding protein (FCaBP) of T. cruzi. Each protein contains an N-terminal glycine followed closely by a cysteine residue, the sites of myristoylation and palmitoylation, respectively. Residues conserved in each protein are shown in bold and underlined. (B) The wild-type Tb44 open reading frame was cloned into the pLEW79-Myc vector for parasite expression with a C-terminal epitope tag. Expression was analyzed by anti-myc immunoblotting of 5x106 cell equivalents of lysate. The wild-type protein underwent myristoylation and palmitoylation, as detected by metabolic labeling and acyl-biotin exchange chemistry, respectively. Mutations were introduced into the predicted sites of acylation. The G2A mutant lacked both myristoylation and palmitoylation, whereas the C3A mutant was myristoylated but not palmitoylated.

 

Figure 3
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  		Fig. 3. Localization of acylation mutants. Parasites expressing myc-tagged wild-type (WT), G2A (nonacylated) or C3A (myristoylated) calflagin Tb44 were examined by differential interference contrast (DIC) microscopy and calflagin (myc)-specific (green) or paraflagellar rod (ROD-1)-specific (red) immunofluorescence microscopy. Bar, 5 µm. The wild-type protein is flagellar, the G2A mutant protein is found throughout the cell and the C3A mutant localizes to the pellicular membrane.

 

Figure 4
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  		Fig. 4. Mutations that abolish palmitoylation also disrupt association with lipid rafts. (A) Parasites were fractionated into supernatant (S) and pellet (P) fractions in PBS + 1% Triton X-100 at either 4°C or 37°C as described in the Materials and Methods. The wild-type dually acylated Tb44 protein, but neither acylation mutant, exhibited temperature-dependent detergent resistance. (B) Parasite extracts were loaded at the bottom (Fraction 10) of a discontinuous Optiprep density gradient and subjected to ultracentrifugation. Fractions were collected and analyzed by anti-myc immunoblotting. Fraction 2 contains the lipid raft interface. The wild-type dually acylated Tb44 protein, but neither acylation mutant, floated to the lipid raft interface.

 

Figure 5
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  		Fig. 5. Calflagins localize to the pellicular membrane upon inhibition of TbPAT7. RNAi against each candidate PAT was induced by the addition of tetracycline to cultured procyclic cells. Calflagin immunofluorescence microscopy was obtained 48 hours post-induction. (A) Representative cells of each mutant are shown, with the number in the top left corner of each box indicating the target TbPAT. Inhibition of TbPATs 1-6 and 8-12 had no effect on the flagellar localization of calflagin, whereas TbPAT7 RNAi resulted in calflagin localization to the pellicular membrane. Bar, 5 µm. (B) The TbPAT7 RNAi cell is shown at higher magnification together with DIC microscopy. Close inspection reveals that fluorescence is restricted to the pelliculum, with no fluorescence in the flagellum (outlined, lower right).

 

Figure 6
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  		Fig. 6. Quantification and kinetics of calflagin mislocalization upon TbPAT7 RNAi. (A) The parental wild-type and all the PAT mutants were analyzed 48 hours post-induction of RNA interference by determining the proportion of cells showing flagellar localization of calflagins. Calflagin mislocalization was observed in ~95% of TbPAT7 RNAi cells but in no more than 5% of any other cell. (B) The proportion of TbPAT7 mutant cells showing flagellar localization of calflagins was analyzed during a time course of RNAi induction and release. At the indicated times post-induction, parasites were removed from the culture and fixed with paraformaldehyde. After 96 hours, tetracycline was washed out by three sequential rounds of centrifugation and resuspension in PBS (first two spins) or fresh medium. At the conclusion of the time course, all slides were analyzed by calflagin immunofluorescence microscopy. Calflagin mislocalization exhibited a t1/2 of ~24 hours and was reversible upon tetracycline washout. Error bars indicate 95% confidence intervals. Between 40 and 100 individual cells were scored at each data point by calflagin immunofluorescence microscopy.

 

Figure 7
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  		Fig. 7. Depletion of TbPAT7 inhibits calflagin palmitoylation. Lysates were harvested from wild-type (WT) and TbPAT7 mutant cells ({Delta}7) that were induced for RNAi for 48 hours and analyzed by acyl-biotin exchange and calflagin immunoblotting. The input material from Tris- and NH2OH-treated samples is shown on the left and the streptavidin eluates from acyl-biotin exchange reactions are shown on the right. Only in wild-type cells are the calflagins palmitoylated, as determined by their ability to be biotinylated by the acyl-biotin exchange reaction in a NH2OH-dependent manner, and subsequently purified by streptavidin and detected by immunoblotting with calflagin-specific antiserum.

 

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