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First published online 24 October 2006
doi: 10.1242/jcs.03280

Journal of Cell Science 119, 4678-4688 (2006)
Published by The Company of Biologists 2006
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Cellular uptake of fatty acids driven by the ER-localized acyl-CoA synthetase FATP4

Katrin Milger1, Thomas Herrmann1, Christiane Becker1, Daniel Gotthardt1, Jelena Zickwolf1, Robert Ehehalt1, Paul A. Watkins2, Wolfgang Stremmel1,* and Joachim Füllekrug1,*,{ddagger}

1 Department of Gastroenterology, Im Neuenheimer Feld 345, University of Heidelberg, 69120 Heidelberg, Germany
2 Kennedy Krieger Institute, 707 N. Broadway, Baltimore, MD 21205, USA


Figure 1
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  		Fig. 1. Intracellular localization of FATP4-GFP. (A) Intracellular reticular localization of FATP4 in stably expressing MDCK cells. Shown is a single section obtained by confocal microscopy to demonstrate the reticular distribution clearly. (B) FATP4 colocalization with the ER marker protein CaBP1 in transiently transfected HeLa cells. (C) FATP4 comparison with a plasma membrane marker protein (CD8); transiently expressing HeLa cells. Only one confocal plane close to the bottom is shown. Bars, 10 µm. (D,E) Intracellular localization of FATP4 in terminally polarized MDCK cells. (D) The apical plasma membrane (side view) is stained with an antibody to gp114. (E) The lateral plasma membrane (midsection, one confocal plane only) is identified by p58. Note that FATP4-GFP is present between the plasma membrane and the nucleus, and not colocalizing with p58. Bars, 10 µm.

 

Figure 2
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  		Fig. 2. Localization of wt FATP4. (A) Expression of FATP4 in COS cells. Lysozyme-KDEL (green) was cotransfected as an ER marker protein. Staining of the nuclear envelope and the reticular network-like pattern (inset) indicates ER localization. Both proteins clearly label the same structure but there is some microheterogeneity. Inhibition of protein synthesis using cycloheximide did not change the localization pattern of FATP4. Shown is a single confocal plane. Bar, 10 µm. (B) Coexpression of FATP4 and a plasma membrane marker protein (ICAM-1-GFP) in Vero cells. There is no significant overlap, which is especially evident at the edge shown in the magnified inset. Bar, 10 µm. (C) Codistribution of endogenous FATP4 with ER membranes after subcellular fractionation. A postnuclear supernatant was prepared from HeLa cells and analyzed by velocity-controlled density centrifugation on an Optiprep step gradient. Fractions were collected from top to bottom and analyzed by SDS-PAGE and western blotting. Calnexin and Na+K+-ATPase served as markers for the ER and the plasma membrane, respectively.

 

Figure 3
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  		Fig. 3. Immunohistochemistry of mouse small intestine. (A) Overview of FATP4 distribution. FATP4 (green) is mostly above the nuclei (blue) and oriented towards the lumen of the gut. Intestinal villi are shown in midsection. Bar, 40 µm. (B) Comparison of FATP4 and an apical marker protein. Sections were labeled with affinity purified antibodies against FATP4 and a mouse monoclonal antibody against PLAP. Pre-incubation with the C-terminal peptide of FATP4 abolishes the cytoplasmic staining pattern of FATP4. Exposure times for sections were identical. Bar, 10 µm. (C) Analysis of intestinal lysates of embryos of wt and FATP4 KO mice. Equal amounts of total lysate (50 µg) were separated by SDS-PAGE and developed by western blotting. A strong band at 72 kDa indicates the presence of FATP4 in the wt but the absence in the intestine of KO mice. Molecular mass markers are indicated in kDa.

 

Figure 4
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  		Fig. 4. Topology of FATP4. (A) Glycosylation analysis. HeLa cells were transfected with N-terminal-tagged FATP4 variants containing either consensus sites for N-glycosylation (opsinF4) or not (ops-ctrl). Membrane preparations were treated with EndoH as indicated. The upper band in the third lane marks an EndoH-sensitive glycosylation, suggesting that opsinF4 is restricted to the ER. (B) Proposed topology for FATP4. The N-terminus is located in the lumen of the ER. A single TMD is followed by the ERx domain. The acyl-CoA synthetase homology region (ACS) corresponds to the protein family of AMP-binding enzymes (pfam00501). (C) Overview of FATP4 mutant proteins. OpsinF4 contains an N-terminal extension allowing N-glycosylation. Ops-ctrl has two amino acid changes destroying the consensus sites for glycosylation. FLAG-FATP4 features an N-terminal epitope tag. S247A contains an inactivating point mutation in the AMP-binding region; serine 247 is changed to alanine. (D) Surface quantification of FLAG-FATP4 by FACS analysis. COS cells were transfected with FLAG-FATP4 or the control plasmid pcDNA3, and processed for FACS analysis either PFA fixed and TX-100 permeabilized (sample 1) or not permeabilized (samples 2, 3). The percentage of gated cells was multiplied with the geometric mean of the fluorescence signal derived from the FLAG antibodies to give the arbitrary value for the total signal (SFLAG). (1) Expression of FLAG-FATP4 and permeabilization with TX-100 yields the maximum FLAG signal. (2) Cells transfected with pcDNA3 do not express the FLAG epitope; the remaining signal is because of unspecific binding. The FATP4 antibody gives a signal for endogenous protein. (3a) Cells transfected with FLAG-FATP4, not permeabilized. This signal is in the background range, and the cells were also positive for the internal antigen FATP4, revealing that the surface is leaky. (3b) This is a subpopulation of 3a and would represent cells with an intact surface that are positive for the FLAG signal but negative for FATP4. See also the Materials and Methods section for more details.

 

Figure 5
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  		Fig. 5. Targeting of FATP4. (A) Domain architecture of GFP reporter proteins. F4-Nt-GFP contains the first 102 amino acids of mouse FATP4 followed by GFP. LAT-GFP comprises the N-terminal targeting domain (extracellular part, orange box; TMD, light-blue box) of LAT fused to GFP. LAT-ERx-GFP contains the ERx domain of FATP4 (amino acids 47-102, yellow box) inserted into LAT-GFP. (B) The N-terminus of FATP4 is sufficient for the ER localization. Vero cells were transiently transfected with plasmid F4-Nt-GFP and treated for 2 hours with the protein synthesis inhibitor cycloheximide before fixation. Localization to the ER is evidenced by co-distribution with the marker protein lysozyme-myc-KDEL. (C-E) Targeting mediated by the ERx domain. The reporter protein LAT-GFP is localized to the plasma membrane (C). (D,E) Insertion of the 56 amino acids of the ERx domain is sufficient to retain the corresponding LAT-ERx-GFP protein intracellularly. LAT-ERx-GFP is present in the ER (co-distribution with lysozyme-myc-KDEL; D) and in a perinuclear compartment, clearly different from the plasma membrane marked by PLAP (E). Bar, 10 µm.

 

Figure 6
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  		Fig. 6. Localization and targeting of ACSL1. (A) Epitope-tagged ACSL1 was co-expressed with a mitochondrial marker protein (OCT-GFP) in Ptk2 cells. One representative section obtained by confocal microscopy is shown. Bar, 10 µm. (B) A1-Nt-GFP contains the 66 N-terminal amino acids of ACSL1 followed by GFP. Overlap with a mitochondrial red fluorescent protein (RFP) coexpressed in Vero cells demonstrates that the N-terminus of ACSL1 is sufficient for targeting. Bar, 10 µm.

 

Figure 7
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  		Fig. 7. Simultaneous analysis of expression, localization and fatty acid uptake. FATP4-(A) or ACSL1 (B)-transfected COS cells were incubated for 2 minutes with 20 µM of the fluorescent fatty acid analog B12-FA, fixed and processed for indirect immunofluorescence. Untransfected cells (outlined, nuclei marked with an asterisk) indicate the background level of fluorescent fatty acid uptake. The distinct reticular ER pattern of FATP4 demonstrates that intracellular localization is sufficient to drive enhanced uptake of fatty acids. Cells expressing ACSL1 localized to mitochondria also show higher B12-FA uptake than untransfected cells. Bar, 10 µm.

 

Figure 8
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  		Fig. 8. Analysis of fatty acid uptake. (A) FACS of COS cells cotransfected with the red fluorescent tdimer protein. This enables the correlation of expression with the amount of the fluorescent fatty acid analog B12-FA taken up. Both FATP4 (r=0.93) and ACSL1 (r=0.71) enhance fatty acid uptake depending on their relative level of expression. The S247A mutant FATP4 protein (serine 247 critical for AMP binding changed to alanine) is not significantly different from control cells expressing only tdimer. (B) Acyl-CoA synthetase activity (in pmol oleoyl-CoA/minute/µg protein) determined from the lysates of COS cells. Controls for endogenous ACS activity are transfected with empty plasmid (pcDNA3) or the S247A-FATP4 mutant. The ACS activities should be considered qualitative rather than absolute because ACSL1 and FATP4 showed inverse susceptibilities towards the detergent used for solubilization (see Materials and Methods). (C) Oleate uptake of COS cells (pmol oleate/µg protein after 5 minutes). (D) Lipid analysis after oleate uptake. After 5 minutes, 87-92% of oleate is already metabolized into phospholipids and neutral lipids. The level of the remaining free oleate is only 5-8%.

 

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© The Company of Biologists Ltd 2006