PEX16 localizes to peroxisomes and ER and is not present in cytosol
To characterize the dynamic distribution of human PEX16, the COOH terminus of PEX16 was tagged with monomeric versions of various fluorescent proteins (GFP, photoactivatable GFP [PAGFP], or Venus). Two lines of evidence suggested that all of the resulting chimeras targeted and functioned properly when expressed in mammalian cells. First, when PEX16 tagged with GFP (PEX16-GFP) was expressed in COS-7 cells, complete colocalization was observed between PEX16-GFP and a coexpressed peroxisomal reporter molecule consisting of the red fluorescent protein (RFP) tagged to type 1 peroxisomal matrix targeting signal, SKL-COOH (RFP-SKL; ). Second, in cells from the human fibroblast cell line GM06231 lacking peroxisomes because of a mutated PEX16 gene, introduction of PEX16-GFP led to the appearance of new peroxisomes (Brocard et al., 2005
), indicating PEX16-GFP can complement PEX16 function.
Figure 1. PEX16-GFP localizes to both peroxisomes and ER. (A) COS-7 cells transiently coexpressing PEX16-GFP and RFP-SKL imaged 15 h after transfection. (B) COS-7 cells coexpressing PEX16-GFP and ssRFP-KDEL imaged 24 h after transfection. (C) Same as A except the (more ...)
COS-7 cells expressing PEX16-GFP at higher levels, achieved by increasing the time between cell transfection and imaging (from 15 to 24 h; Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200601036/DC1
), were examined to determine whether PEX16-GFP changed its distribution once the machinery involved in PEX16 sorting to peroxisomes became limited by PEX16 overexpression. To identify peroxisomes as well as ER in these cells, the peroxisomal marker RFP-SKL and the ER marker ssRFP-KDEL were individually coexpressed with PEX16-GFP. The distribution of PEX16-GFP in these cells included the ER as well as peroxisomes (). Hence, the membrane localization of PEX16-GFP is not restricted to peroxisomes but includes the ER under conditions of high PEX16-GFP expression.
Expression of PEX16-GFP at low levels in the peroxisome biogenesis disorder (PBD) 399-T1 human fibroblast cell line that lacks PEX19 and in which peroxisomes are absent (Sacksteder et al., 2000
) showed the chimera residing exclusively in the ER, with RFP-SKL diffusely distributed throughout the cytosol (). A similar ER pattern was observed for PEX16-GFP expressed in a PEX3 mutant human fibroblast cell line (PBD400) that, similar to PBD399-T1 cells, lacks peroxisomes (South et al., 2000
; unpublished data). When peroxisomes are absent, therefore, PEX16-GFP targets to the ER and not to the cytosol. Cell fractionation and immunoblot analysis of PEX16-GFP–transformed COS-7 cells performed 24 h after transfection to allow for protein overexpression revealed that PEX16-GFP resided only in the membrane (or nonsoluble fraction), in contrast to GFP expressed alone, which was primarily in the soluble fraction (). Hence, PEX16-GFP does not appear to ever reside in the cytosol.
Peroxisomes that were observed in cells having high PEX16-GFP expression were closely aligned with the ER ( [box] and D), suggesting that peroxisomes and the ER are intimately associated. Repetitive photobleaching (or fluorescence loss in photobleaching [FLIP]) of PEX16-GFP fluorescence in a small, centralized area of the ER in these cells (, red box) resulted in PEX16-GFP fluorescence being lost throughout the ER without affecting PEX16-GFP fluorescence in surrounding peroxisomes (). Molecules of PEX16-GFP can thus freely diffuse throughout the ER, whereas they are retained within individual peroxisomes.
Photoactivated PEX16-PAGFP moves from the ER to peroxisomes
To investigate whether the ER localization of PEX16 represented an intermediate in the pathway for delivery of PEX16 to peroxisomes, we developed a photo/pulse-chase–labeling assay using PEX16 attached to PAGFP (PEX16-PAGFP). PAGFP is undetected until “activated” by high-energy light, whereupon it becomes brightly fluorescent. Activated PAGFP molecules remain fluorescent over time, whereas PAGFP molecules that have not been photoactivated (including newly synthesized and newly folded forms) stay invisible (Patterson and Lippincott-Schwartz, 2002
The photo/pulse-chase assay was performed in COS-7 cells coexpressing PEX16-PAGFP and RFP-SKL 24 h after transfection, as outlined in . Initially, a small region of interest (ROI; , red box) containing only ER (blue) and no peroxisomes (red) was repeatedly irradiated over 30 min with 413-nm light (pre-PA). Because PEX16-GFP diffuses freely throughout the ER, most PEX16-PAGFP molecules in the ER should become photoactivated (, green) under this treatment. An image of the cell was collected sequentially in the 543-nm channel to visualize peroxisomes containing RFP-SKL immediately before and after each photoactivation to ensure no peroxisomes moved into the ROI and that no PEX16-PAGFP molecules within peroxisomes become photoactivated. After photoactivation in this manner for 30 min, images of PEX16-PAGFP at 488-nm fluorescence and RFP-SKL at 543-nm fluorescence were acquired (, Post-PA t = 0 min). The cell was then incubated for 5 h (chase) before acquiring another set of images (, Post-PA t = 5 h) to assess whether fluorescent PEX16-PAGFP molecules had redistributed to RFP-SKL–containing peroxisomes.
Figure 2. Photo/pulse-chase assay indicates that PEX16-PAGFP, but not delPEX16-GFP, sorts from the ER to peroxisomes. (A) Schematic diagram of the photo/pulse-chase assay. (B) Photo/pulse-chase assay performed in COS-7 cell coexpressing PEX16-PAGFP and RFP-SKL. (more ...)
As shown in , before photoactivation of the ER pool of PEX16-PAGFP, GFP fluorescence (excited at 488 nm) was negligible, whereas the fluorescence attributable to RFP-SKL (543 nm) was readily visible and localized primarily to individual (punctate) peroxisomes (, Pre-PA). Upon repeated photoactivation of the small ROI in the lower left part of the cell, PEX16-PAGFP fluorescence became visible in the ER and in a few puncta that partially overlapped with RFP-SKL, suggesting they were peroxisomes (, Post-PA t = 0 min, arrows). After the 5-h chase period, significantly more PEX16-PAGFP fluorescence was localized in peroxisomes (, Post-PA t = 5 h, arrows). Quantification of peroxisomes containing both PEX16-PAGFP and RFP-SKL over the 5-h chase period in five independent experiments revealed that 10–40% of all peroxisomes in these cells became labeled with photoactivated PEX16-PAGFP (unpublished data). Because PEX16-PAGFP molecules were pulse-labeled in the ER and later appeared in peroxisomes, the data suggested that PEX16-PAGFP undergoes specific transport from the ER to peroxisomes.
PEX16 movement from ER to peroxisomes depends on sequences in its NH2 terminus
To investigate what molecular features of PEX16 were necessary for it to pass from the ER to peroxisomes, we used a PAGFP-tagged variant of PEX16 in which the NH2
-terminal membrane peroxisome–targeting sequence (residues 66–81; -RKELRKKLPVSLSQQK-; Honsho et al., 2002
) was deleted. Expression of this construct (delPEX16-PAGFP) in COS-7 cells resulted in only an ER pattern of localization with no peroxisome labeling (Fig. S2, available at http://www.jcb.org/cgi/content/full/jcb.200601036/DC1
). In the photo-chase assay, photoactivation of the ER pool of a cell expressing delPEX16-PAGFP resulted in the fluorescence attributable to delPEX16-PAGFP never redistributing to peroxisomes and remaining within the ER (). The small structures containing photoactivated delPEX16-PAGFP seen in the juxtanuclear region of these cells (, Post-PA t = 0 min and Post-PA t = 5 h) presumably represented compacted ER cisternae, as their fluorescence was diminished upon repeated photobleaching of a small area of ER in delPEX16-GFP–expressing cells (Fig. S2). Thus, delivery of PEX16 from ER to peroxisomes is dependent on the membrane peroxisome–targeting sequence found within PEX16.
PEX16 with an appended NH2-terminal type I signal anchor sequence targets to peroxisomes after being synthesized in the ER
Nascent polypeptides containing an NH2
-terminal signal sequence are bound by the signal recognition particle in the cytoplasm and transferred to the ER before the remainder of their mRNA is translated (Rapoport et al., 1996
). By attaching such a sequence to the NH2
terminus of PEX16, we reasoned that we could force newly synthesized PEX16 proteins to be cotranslationally inserted into the ER before they targeted elsewhere in the cell (such as to peroxisomes). With such a construct, we could then address whether PEX16 could target to peroxisomes after being cotranslationally synthesized in the ER. We appended residues 14–90 from the well-defined type I signal anchor sequence of leader peptidase (designated as sa; Gafvelin et al., 1997
; Heinrich et al., 2000
) to the NH2
terminus of PEX16-GFP (yielding saPEX16-GFP) to preserve the native (Nout
; ) membrane topology of PEX16 (Honsho et al., 2002
). Evidence that sa functioned properly as a signal sequence for targeting of proteins to the ER was demonstrated by appending it to GFP alone (producing saGFP) and expressing the construct in COS-7 cells treated with brefeldin A to block secretory transport out of the ER. In these cells, saGFP accumulated in the ER (), whereas untagged GFP expressed in COS-7 cells was distributed diffusely throughout the cytosol and nucleus ().
Figure 3. PEX16 containing an NH2-terminal signal-anchor sequence localizes to both peroxisomes and ER. (A) Illustration of the predicted topologies of PEX16-GFP, saPEX16-GFP, and saGFP. sa sequences are shown in red, PEX16 in black, and GFP in green. The asterisk (more ...)
To test whether saPEX16-GFP could target to peroxisomes after biosynthesis in the ER, we examined COS-7 cells coexpressing saPEX16-GFP and ssRFP-KDEL 15 h after transfection. A large pool of saPEX16-GFP could be seen colocalized with ssRFP-KDEL in the ER (). Differential permeabilization and antibody binding experiments demonstrated that saPEX16-GFP in the ER maintained the same Cout
topology as that of PEX16-GFP (Fig. S3, available at http://www.jcb.org/cgi/content/full/jcb.200601036/DC1
). Repetitive photobleaching (i.e., FLIP) of saPEX16-GFP fluorescence in an area of ER abolished saPEX16-GFP fluorescence throughout the ER and revealed a pool of saPEX16-GFP fluorescence in peroxisomes (; note the localization of saPEX16-GFP– in RFP-SKL–containing peroxisomes in ). Therefore, saPEX16-GFP can undergo transport to peroxisomes after being synthesized in the ER.
When the cells were imaged at earlier times after transfection (8 h) to examine saPEX16-GFP localization at lower expression levels, saPEX16-GFP was exclusively colocalized with RFP-SKL in peroxisomes (). A possible explanation for this is that saPEX16-GFP moves efficiently from ER to peroxisomes until excess saPEX16-GFP saturates the machinery for targeting to peroxisomes. Consistent with this, time-lapse imaging of cells expressing saPEX16-GFP over a 10-h period (during which saPEX16-GFP expression levels went from low to high) revealed newly synthesized saPEX16-GFP accumulating in peroxisomes before also accumulating in the ER ().
PEX16 with a signal anchor sequence complements wild-type PEX16 in cells lacking the PEX16 gene
To determine whether saPEX16-GFP could complement PEX16 function and give rise to new peroxisomes, cells from the human GM06231 cell line lacking peroxisomes were transfected with RFP-SKL, PEX16-GFP and RFP-SKL, or saPEX16-GFP and RFP-SKL. The distribution of GFP and/or RFP fluorescence in these cells was then examined 48 h after transfection (). In cells transfected with RFP-SKL alone, the peroxisomal reporter was distributed diffusely in the cytosol and no fluorescence at 488 nm was detected, indicating that peroxisomes were indeed absent in these cells (). In contrast, in cells cotransfected either with PEX16-GFP and RFP-SKL () or with saPEX16-GFP and RFP-SKL (), numerous punctate and globular peroxisomal structures containing both sets of expressed proteins were observed (, arrows). Because the signal anchor sequence on saPEX16-GFP forced it to be inserted into the ER membrane before delivery to other membranes, these results indicated that a pathway from the ER involving PEX16 was sufficient to support peroxisome production de novo.
Figure 4. saPEX16- and PEX16-GFP rescue PEX16-deficient GM06231 cells. (A–C) GM06231 cells were transiently transfected with pRFP-SKL alone (A) or cotransfected with RFP-SKL and either PEX16-GFP (B) or saPEX16-GFP (C). Live cells were imaged 48 h after (more ...)
Interestingly, some of the punctate structures in the cells coexpressing the GFP-tagged PEX16 and RFP-SKL molecules contained only PEX16- or saPEX16-GFP. These structures may represent so-called early or nascent peroxisomes (South and Gould, 1999
; Honsho et al., 2002
) that have not yet begun importing lumenal peroxisomal proteins after PEX16
Wild-type PEX16 inserts into ER membranes in a cotranslational, rather than posttranslational, manner in vitro
There are two ways in which wild-type nascent PEX16 can target to the ER: (1) by posttranslational targeting, in which PEX16 is synthesized on free ribosomes in the cytoplasm and then is posttranslationally targeted to and inserted into ER membranes, or (2) by cotranslational targeting, in which ribosomes containing NH2
-terminal PEX16-nascent chains are first targeted to the ER and then PEX16 is cotranslationally inserted into ER membranes. To distinguish between these two possibilities, we used an in vitro binding assay in which PEX16 was synthesized using a rabbit reticulocyte lysate in the presences of 35
S-methionine. To assay for cotranslational targeting, the translation reaction was performed in the presence of ER microsomes. To assay for posttranslational targeting, ER microsomes were added to the reaction after the protein was first fully translated and further protein translation was inhibited by the addition of cycloheximide. Preprolactin (PPL) and cytochrome b5
-glyc; cytochrome b5
with a glycosylation site in its luminal domain) were used as appropriate cotranslation and posttranslation controls, respectively (Andrews et al., 1989
; Pedrazzini et al., 2000
As shown in , PEX16 pelleted more readily with ER microsomes during cotranslational targeting (39%) than during posttranslational targeting (18%) or during translation without microsomes (10%). These results were similar to those observed for the cotranslational targeting of PPL; i.e., significantly more PPL pelleted with microsomes with its signal sequence cleaved during co-T2 targeting compared to post-T2 targeting ( [PPL, compare lane 3 with 6 and 9] and B). In contrast, Cb5
-glyc exhibited binding to ER microsomes both when microsomes were added during the translation reaction and when microsomes were added after translation (, Cb5
-glyc), as expected for this posttranslationally targeted protein (Pedrazzini et al., 2000
). Furthermore, the actual integration of Cb5
-glyc into ER microsomes was demonstrated by its glycosylation, which shifted it to a higher molecular mass in the pellet fraction (, lanes 3 and 6).
Figure 5. PEX16 targets cotranslationally to ER in vitro. (A) Autoradiographs of the in vitro ER microsome binding assays with PPL (~30 kD), Cb5-glyc (~20 kD), and PEX16 (~37 kD) in both cotranslational (co-T2) targeting and posttranslational (more ...)
The increase of PEX16 in the pellet fraction during posttranslational targeting experiments compared with minus membrane experiments () may be due to nonspecific interactions of PEX16's hydrophobic transmembrane domains with microsomes rather than to PEX16's actual integration into the lipid bilayer of the ER. To determine whether this was the case, we engineered a glycosylation site at the putative luminal domain of PEX16 (PEX16-glyc; N-X-S starting at residue 161; ). We reasoned that if any PEX16-glyc molecules in the assay were glycosylated, they must have been specifically integrated into ER microsomes. As shown in , a significant proportion of PEX16-glyc molecules underwent glycosylation during cotranslational targeting, whereas none did so during posttranslational targeting. The data thus confirmed that PEX16 undergoes cotranslational insertion into the ER under in vitro conditions.
PEX16 can recruit other PMPs to the ER
It was previously hypothesized that PEX16 acts as part of the machinery involved in recruiting PMPs to membranes (Honsho et al., 2002
; Fang et al., 2004
). If so, overexpressed PEX16 that is localized to the ER should cause other PMPs, such as PEX3 and PMP34, to retarget to ER membranes. To test this prediction, we constructed chimeras of PEX3 and PMP34 fused to the GFP or Cerulean blue fluorescent protein (Rizzo et al., 2004
) and examined their subcellular location in the presence or absence of overexpressed PEX16 fused to the Venus fluorescent protein (PEX16-Venus; Nagai et al., 2002
Neither PEX3- nor PMP34-GFP was targeted to the ER when expressed in cells lacking peroxisomes (Fig. S4, available at http://www.jcb.org/cgi/content/full/jcb.200601036/DC1
) or in cells containing peroxisomes (). Indeed, PEX3-GFP was colocalized exclusively with RFP-SKL in peroxisomes at low expression levels () and accumulated in mitochondria at higher expression levels (). PMP34-Cerulean was also localized to peroxisomes at low expression levels () but accumulated in the cytoplasm at higher expression levels (). Importantly, when PEX16-Venus was coexpressed with either PEX3- or PMP34-Cerulean, both PMPs colocalized with PEX16-Venus in the ER (). When a small area of the ER was repeatedly photobleached to remove ER fluorescence in cells coexpressing the PMPs and PEX16-Venus, both PMPs were observed in peroxisomes (unpublished data). Similar colocalizations of PEX16-Venus and PEX3-Cerulean in the ER were observed in the PBD399-T1 fibroblast cells lacking peroxisomes (Fig. S4), indicating that the recruitment of PEX3-Cerulean by PEX16-Venus to the ER also occurred in other cells. Hence, PEX16 appears to function in the recruitment of PEX3 and PMP34, and possibly other PMPs, to membranes.
Figure 6. PEX16 recruits PEX3 and PMP34 to the ER. (A and B) COS-7 cells either coexpressing PEX3-GFP and pRFP-SKL 15 h after transfection (A) or expressing PEX3-GFP and stained with MitoTracker 24 h after transfection (B). (C) A COS-7 cell expressing PEX16-Venus (more ...)
Distinguishing de novo peroxisome biogenesis from peroxisome fission in wild-type cells
Recent studies in S. cerevisiae
cells have suggested that de novo biogenesis of peroxisomes occurs by direct outgrowth of peroxisomal structures from the ER (Hoepfner et al., 2005
; Kragt et al., 2005
; Tam et al., 2005
). To test whether a similar mechanism occurs in mammalian cells, we devised an assay to directly visualize the formation of peroxisomes in wild-type mammalian cells (). The assay involved two photoactivation events separated by 24 h that permitted old and new peroxisomal components to be differentiated in living cells. We reasoned that daughter peroxisomes formed by division of preexisting peroxisomes should all contain peroxisomal components from their mother peroxisomes. However, peroxisomes formed by a de novo pathway from the ER should contain only newly synthesized components. Therefore, by distinguishing recently synthesized peroxisomal components from older peroxisomal components, we could distinguish peroxisomes formed de novo from those formed by fission.
Figure 7. Peroxisome biogenesis assay. (A) Schematic representation of the peroxisome photo-chase biogenesis assay. (B) Images of PAGFP-SKL at different time points during the assay. (C) Enlargement of the areas outlined by the stippled boxes in post-1st PA t = (more ...)
NRK cells were transiently transfected with PAGFP fused to SKL (PAGFP-SKL) to mark preexisting peroxisomes and with PEX16-Cerulean to monitor peroxisomes before (pre-PA) and after (post-1st PA t = 0 h) photoactivation of PAGFP-SKL (). All PAGFP-SKL molecules in the peroxisomes of the cell were initially photoactivated using 413-nm laser light, and an image was collected (post-1st PA t = 0 h). The cell was then incubated at 37°C for 24 h to allow newly synthesized, nonphotoactivated PAGFP-SKL molecules to accumulate. The cell was then fixed to prevent peroxisomes from moving, and another image was collected (post-1st PA t = 24 h). Immediately thereafter, the cell was photoactivated a second time (re-PA) to highlight all newly synthesized (previously “invisible”) PAGFP-SKL molecules, and a final image was collected (post-2nd PA). The image acquired after the second PA (post-2nd PA) was compared with the previous image (i.e., post-1st PA t = 24 h) to determine whether the PAGFP-SKL molecules that were synthesized during the 24 h were only localized to previously fluorescent peroxisomes or if they were localized to both previously fluorescent peroxisomes and nonfluorescent nascent peroxisomes within in the cell.
Before photoactivation, no fluorescence attributable to PAGFP-SKL was observed in a cell coexpressing PAGFP-SKL and PEX16-Cerulean (, pre-PA; and not depicted). However, upon photoactivation, punctate peroxisomes containing PAGFP-SKL became brightly fluorescent (, post-1st PA t = 0 h). Imaging of the same cell 24 h later revealed a minimal loss of fluorescent signal in the cell (, post-1st PA t = 24 h). Indeed, quantification of the PAGFP-SKL fluorescence in different cells throughout the 24-h period revealed that the level of fluorescence remained virtually constant (). This suggested that PAGFP-SKL proteins were long-lived and that repeated low-light imaging did not lead to their fluorescence being photobleached. The number of fluorescent peroxisomes measured at the start and end of this imaging period only slightly increased (~8%), presumably because of peroxisomes undergoing fission (, fission).
After the second photoactivation, the fluorescence associated with already fluorescent peroxisomes increased significantly (, compare post-1st PA t = 24 h and post-2nd PA). Once formed, therefore, peroxisomes continue to import newly synthesized PAGFP-SKL. Notably, fluorescence also appeared in peroxisomes that were not previously fluorescent (i.e., peroxisomes that did not contain any PAGFP-SKL molecules highlighted during the first photoactivation step; [post-2nd PA] and C [higher magnification, arrows]). These “new” peroxisomes were not the result of preexisting peroxisomes that had lost their fluorescent signal by either degradation or photobleaching after the initial photoactivation, as the total fluorescent signal from photoactivated PAGFP-SKL did not diminish over the 24-h chase period (). Rather, they appeared to represent peroxisomes formed de novo during the 24-h chase period.
Quantification of the number of newly appearing peroxisomes after the second photoactivation event was determined by calculating the difference in the number of peroxisomes before and after the second photoactivation. A mean of 30 ± 10 new peroxisomes per cell was found based on results from eight independent experiments (). This represented an ~20% increase in the total number of peroxisomes in the cell over the 24-h period and was much greater than the slight increase in peroxisomes observed after the first photoactivation based on a t test (t = 2.42; P < 0.05). Therefore, a de novo pathway appeared to play a significant role in the formation of peroxisomes in these cells.