BACKGROUND
peroxisomes are a diverse group of organelles which accommodate many activities related to lipid metabolism.
in mammals, these organelles harbor a number of essential metabolic functions including ether phospholipid biosynthesis, fatty acid α – and β-oxidation, and glyoxylate detoxification  <cit> .
the size, number and enzyme content of peroxisomes can be affected by various environmental, metabolic, and developmental factors  <cit> .
peroxisomes are thought to arise de novo from the endoplasmic reticulum   <cit>  or multiply by fission of pre-existing peroxisomes  <cit> .
the relative contribution of both pathways to the total number of peroxisomes in wild-type cells is not yet clear  <cit> , and the molecular mechanisms governing these processes are only beginning to be unraveled  <cit> .
studies in different model systems have demonstrated that the pex11p family of peroxisomal membrane proteins , a select set of dynamin-related proteins , and fis <dig> and mff – two putative adaptors for drps – coordinately control the shape, size and number of peroxisomes in a cell  <cit> .
the pex <dig> proteins are supposed to be involved in the elongation and tubulation of pre-existing peroxisomes, whereas the drps, fis <dig>  and mff are thought to catalyze the final fission event  <cit> .
it has been put forward also that adp-ribosylation factors and coatomer proteins are involved in the biosynthetic process of the peroxisomal membrane  <cit> .
arguments in favor of this hypothesis are the observations that  arf <dig>  arf <dig>  and copi coatomer proteins can bind to highly purified rat liver peroxisomes  <cit> ,  expression of a temperature-sensitive ε-cop subunit in cho cells at non-permissive temperature exhibited a dramatic change in peroxisome morphology  <cit> , and  the s. cerevisiae orthologues of mammalian arf <dig>  and arf <dig>  regulate peroxisome division up and down, respectively  <cit> .
also, peroxisomal arf/copi might be involved in the retrieval of cargo from peroxisomes back to the er  <cit> .
the observation that a dominant-negative mutant of arf <dig> inhibits the transport pathway of tomato bushy stunt virus  <dig> kda replication protein from peroxisomes to the er in nicotiana tabacum cells supports this hypothesis  <cit> .
arf <dig> and arf <dig> are – together with sar <dig> – the most comprehensively studied proteins of the adp-ribosylation factor  family  <cit> .
members of this group of small gtpases are believed to control vesicular trafficking and organelle structure by recruiting coat proteins, to function as regulators of phospholipid metabolism, and to modulate cytoskeletal organization  <cit> .
arf <dig> has been shown to function in the gtp-dependent recruitment of copi coatomer to budding transport vesicles that move from the golgi to the er  <cit> .
in addition, it has been shown that arf1-gtp regulates the recruitment of clathrin to late golgi and endosome compartments through binding with heterotetrameric adaptor protein complexes as well as monomeric golgi-localized γ-ear-containing arf-binding proteins  <cit> .
arf <dig>  a protein predominantly associated with the plasma membrane, is thought to regulate endosomal membrane delivery and traffic, membrane raft trafficking, membrane recycling during phagocytosis, and actin remodeling at the cell periphery  <cit> .
in this study, we dissect the potential role of small gtpases, and especially of arf <dig>  in peroxisome biogenesis.
RESULTS
yeast cells deficient in arf <dig> or arf <dig> exhibit a pronounced increase in peroxisome number on oleate compared to wild-type cells
in view of a continuously increasing number of studies reporting that the er may provide peroxisomes with lipids and some membrane proteins in the form of a preperoxisomal structure  <cit> , we investigated whether or not yeast strains deficient in one of the small gtpases that function as a molecular switch in protein trafficking contained functional peroxisomes.
as such, all viable null strains of the rab and the sar1/arf families  <cit>   were tested for their ability to consume oleate as a sole carbon source, a growth condition requiring functional peroxisomes  <cit> .
a wild-type strain and a strain lacking pex5p – the import receptor for peroxisomal matrix proteins containing a c-terminal peroxisomal targeting signal – were included as appropriate controls, and oleate consumption was scored by halo formation.
none of the yeast strains deficient in one of the small gtpases displayed significant changes in halo formation compared to the wild-type strain .
this observation indicates that the corresponding gtpases are not essential for peroxisome biogenesis in yeast.
this conclusion is in line with our observations that none of these strains displayed a detectable peroxisomal sorting defect for the peroxisomal matrix protein marker egfp-pts <dig>  or the peroxisomal membrane protein marker pex11p-egfp .
interestingly, the average number of peroxisomes in oleate-grown cells was substantially increased in the Δarf <dig> and Δarf <dig> strains compared to the wild-type strain .
to determine whether or not these changes were significant, the values obtained for the wild-type and arf-null strains were analyzed more thoroughly.
a one-way anova showed significant differences among the groups .
a subanalysis by the tukey-kramer multiple comparisons procedure revealed that the peroxisome number in oleate-grown Δarf <dig> and Δarf <dig> yeast cells did indeed significantly differ from that in oleate-grown wild-type cells .
by contrast, there was no significant pairwise difference between the peroxisome numbers in oleate-grown Δarf <dig> and wild-type cells .
summarized, these observations indicate that, at least under certain growth conditions, arf <dig> and arf <dig> may be involved in the regulation of peroxisome number in s. cerevisiae.
interestingly, the construction and analysis of a double Δarf1Δarf <dig> deletion mutant revealed a cumulative effect of each individual deletion when the cells were grown on oleate .
copurification of arf proteins with rat liver peroxisomes
in order to investigate whether or not any endogenous arf proteins are associated with mammalian peroxisomes, we probed rat liver subcellular fractions with 1d <dig>  a mouse monoclonal antibody that binds to a linear epitope found in arf proteins but not in other gtp-binding proteins  <cit> .
these experiments revealed the presence of a weak but detectable  <dig> kda 1d9-immunoreactive band in the purified peroxisomal fraction .
by performing membrane floatation experiments and employing arf-specific antibodies, we could show that this protein was membrane-associated and most likely arf <dig> .
consistent with this identification is the observation that antibodies towards arf <dig>  arf <dig>  arf <dig>  and/or arf <dig>  but not arf <dig>  did not yield a signal above background .
note that the anti-arf <dig> antibody employed in this study does not cross-react with arf <dig>  arf <dig>  or arf <dig> , and is only yielding a detectable signal for arf <dig> when this protein is expressed in the high-nanogram to low-microgram range .
arf <dig> was not included in these experiments, as this protein – which is absent in humans – is 96% identical  to arf <dig> .
the results obtained with the anti-arf <dig> antibody were not conclusive.
that is, a very weak signal could be observed, but only after very long exposure times .
however, as the epitope recognized by the monoclonal antibody directed against arf <dig> is unknown, we can not exclude the possibility of some cross-reactivity with arf <dig>  finally, as  subcellular fractionation cannot exclude with absolute certainty that a particular protein is located in a contaminating organelle that co-purifies with the organelle under study, and  the peroxisomal fractions employed also contained small amounts of glutamate dehydrogenase , bip/grp <dig> , and pan-cadherin  , we subjected peroxisomal fractions with different degrees of purity to immunoelectron microscopy.
these experiments established that the anti-arf 1d <dig>  and anti-arf <dig>  antibodies specifically recognized a protein on the outer aspect of the peroxisomal membrane, and not on the membrane of a contaminating organelle.
arf <dig> is not essential for peroxisome biogenesis in mouse hepatocytes
in order to investigate the potential role of arf <dig> in peroxisome biogenesis, we examined the number of peroxisomes, peroxisomal morphology, and the localization of peroxisomal membrane and matrix proteins in arf6-/- fetal hepatocytes  <cit> .
as it is well-known that peroxisomes in cultured hepatoma cells may exhibit distinct alterations of shape, size, and distribution dependent on culture conditions   <cit> , we analyzed high, middle and low density hepatocyte populations from wild-type and knockout animals side-by-side.
probing these cells with antibodies raised against the peroxisomal membrane protein pex14p , the pts <dig> protein catalase , the peroxisomal membrane protein pmp <dig>  or the pts <dig> protein thiolase  showed that ablation of arf <dig> did not visibly alter the localization of peroxisomal proteins in cultured fetal hepatocytes from both control and clofibrate-treated pregnant mice.
clofibrate is a hypolipidemic drug known to induce peroxisome proliferation in rodent liver .
in addition, no evidence could be found for an altered number  or spatial distribution  of peroxisomes.
these findings may be surprising in light of our observations that the number of peroxisomes is altered in s. cerevisiae cells deficient in arf <dig> , the yeast orthologue of mammalian arf <dig>  <cit> .
we suggest that a functional redundancy of different arf proteins may protect peroxisome biogenesis in mammals.
in this context it is interesting to point out that every combination of double – but not single – knockdowns of arf <dig>  arf <dig>  arf <dig> and arf <dig> yielded a distinct pattern of defects in secretory and endocytic traffic in hela cells  <cit> .
co-overexpression of arf <dig> and arf <dig> variants impairs peroxisomal protein import in mammalian cells
as a previous study has shown that exogenously added arf <dig> can bind to highly purified rat liver peroxisomes  <cit> , we investigated whether or not overexpression of wild-type, gtp hydrolysis-defective or  gtp binding-defective variants of arf <dig>  and arf <dig> , individually or in pairwise combinations, affected peroxisomal protein import in mammalian cells.
these experiments demonstrated that overexpression of individual arf variants had no effect on the localization of egfp-pts <dig>  a peroxisomal matrix protein reporter .
however, when arf <dig> and arf <dig> variants were simultaneously expressed, all combinations yielded a mislocalization of the reporter protein to various degrees .
interestingly, co-expression of the gdp-bound dominant-negative mutants arf6t27n and arf1t31n resulted in a partial to complete cytosolic localization of egfp-pts <dig> in approximately 80% of the cells ).
similar findings were observed for hspmp34-myc-his, a pmp reporter protein .
this observation indicates that the mislocalization of egfp-pts <dig> is most likely the indirect result of a peroxisomal membrane protein import deficiency.
at first sight, this hypothesis seems to be at odds with the observation that a simultaneous expression of arf6t27n and arf1t31n did not have a visible effect on the subcellular localization of endogenous pex14p .
however, as  the half-life of peroxisomes in cultured mammalian cells is approximately two days, and  the turnover rate of pex14p is rather low  <cit> , the pex14p signals observed in figure 7b most likely represent pre-existing pex14p molecules which were already localized to peroxisomes before arf6t27n and arf1t31n were co-overexpressed.
interestingly, upon closer inspection, we found that the staining pattern of pex14p was slightly changed in at least some cells co-overexpressing arf6t27n and arf1t31n .
indeed, a diffuse 'background' staining could be observed , and – upon a drastic enlargement of the images – it became also clear that the appearance of the pex14p-immunoreactive particles was affected .
the precise nature and mechanism of these changes remain to be determined.
importantly, arf <dig>  arf <dig>  and arf <dig> could not substitute for arf <dig> in these assays .
in addition, co-expression of the gdp-bound dominant-negative variants of arf <dig> and arf <dig> did not interfere with the sorting of hslk2-myc-his, a mitochondrial reporter protein .
discussion
in many instances, the formation of membrane-delimited compartments from pre-existing organelles is regulated by small gtpases of the rab and arf family  <cit> .
a growing amount of evidence suggests that monomeric gtp-binding proteins may also play a role in peroxisome biogenesis.
for example, mass spectrometry-based quantitative proteomic profiling studies have shown that in yeast rho <dig> is recruited to peroxisomes upon their induction by oleate, and it has been suggested that this protein regulates the assembly state of actin on the peroxisome membrane, thereby controlling peroxisome membrane dynamics and biogenesis  <cit> .
recently, it has also been shown that mya <dig>  an isoform of arabidopsis thaliana myosin xi, is targeted to peroxisomes through an interaction with atrab2ca  <cit> .
furthermore, mammalian arf <dig> and arf <dig> have been shown to bind to isolated rat liver peroxisomes, and scarf <dig> and scarf <dig>  the yeast orthologues of these proteins, have been implicated in the control of peroxisome proliferation  <cit> .
a similar function has been suggested for trypanosoma brucei arf <dig>  a protein which shares characteristics with both arf <dig> and arf <dig> and has a vital role in the maintenance of the endocytic pathway  <cit> .
interestingly, no arf proteins have been identified in proteome studies of yeast and mammalian peroxisomes  <cit> .
a possible explanation might be that the relative abundance of these proteins in the analyzed samples is extremely low.
in this study, we initially investigated whether or not yeast strains deficient in arf <dig>  arf <dig>  arf <dig>  arl <dig>  cin <dig>  vps <dig>  ypt <dig>  ypt <dig>  ypt <dig>  ypt <dig>  ypt <dig>  ypt <dig>  ypt <dig> or ypt <dig> were capable of growing on oleate as a sole carbon source and/or displayed a peroxisomal protein sorting defect.
the results of these experiments demonstrated that all these strains, which embody every viable yeast deletion mutant of the rab and sar1/arf subfamilies of small gtpases, contain functional peroxisomes.
interestingly, the average number of peroxisomes in oleate-grown cells was significantly upregulated in the Δarf <dig> and Δarf <dig> strains compared to the wild-type strain, and this phenotype was even more pronounced in the double Δarf1Δarf <dig> deletion mutant.
this observation indicates that scarf <dig> and scarf <dig> dampen peroxisome proliferation, either directly or indirectly.
although this result confirms previous observations that scarf <dig> and scarf <dig> are implicated in the control of peroxisome proliferation  <cit> , it is not consistent with the authors' conclusion that scarf <dig> is required for the oleate-induced peroxisome proliferation in s. cerevisiae.
the reasons for the discrepancy between our results and the results previously presented might be several.
for example, while we were making use of an arf <dig> deletion mutant, lay et al.
<cit>  utilized an arf <dig> temperature-sensitive mutant in an Δarf <dig> background.
also, while these authors analyzed the peroxisome number per cell after  <dig> hours of growth on oleate, we analyzed the cells after  <dig> or  <dig> days of growth on oleate.
note that after transfer of the cells to oleate-containing medium, the number of peroxisomes per cell continuously increased as a function of time for at least  <dig> days, in the Δarf <dig> and Δarf <dig> strains compared to the wild-type strain .
at this point, it is not clear how arf <dig> and arf <dig> suppress oleate-induced peroxisome proliferation in s. cerevisiae.
one possibility might be that these proteins are involved in the generation of peroxisome-derived vesicles, which transport er-derived factors from peroxisomes back to the er  <cit>  or deliver metabolites, lipids, or  proteins to mitochondria  <cit> .
disturbances in such pathways would result in an increase in peroxisome number.
we next investigated whether or not endogenous arf proteins were associated with the membrane of rat liver peroxisomes.
in a first series of experiments, we obtained evidence that 1d <dig>  a mouse monoclonal antibody with specificity for mammalian arf <dig>  arf <dig>  arf <dig>  arf <dig>  arf <dig>  and – to a lesser extent – arf <dig>  <cit> , recognized a  <dig> kda band in highly purified peroxisomal membrane preparations.
we could show that this  <dig> kda protein was associated with the outside face of the peroxisomal membrane , and identify this protein as arf <dig> .
this protein, which is considered to be the mammalian orthologue of yeast arf <dig>  has been reported to act in a wide range of processes, including endocytosis, cytokinesis, phagocytosis, and the organization of the actin cytoskeleton  <cit> .
our observation that endogenous arf <dig> is also associated with peroxisomes may seem surprising given that the subcellular localization of this protein has already been documented in several reports  <cit> .
however, as  peroxisomes occupy less than 2% of the cell volume  <cit> , a partial  association of arf <dig> with these organelles may have been easily missed.
also, as the strength of the signal and its detection depend heavily on the amount of protein and may be easily masked by stronger signals, the methods employed might not be sensitive enough.
no evidence could be obtained for the association of any other endogenous arf protein with the peroxisomal membrane.
however, these results should be considered with caution as such proteins may dissociate during the purification procedure and be present on the peroxisomal membrane in concentrations below the detection limits of the employed arf-specific antibodies.
to study the function of arf <dig> in peroxisome biogenesis, we first examined the number of peroxisomes, peroxisomal morphology, and the location of  peroxisomal membrane and matrix proteins in  ptk <dig> cells transiently overexpressing either wild-type arf <dig> or a mutant defective in gtp-binding  or gtp-hydrolysis  and  arf6-/- fetal mouse hepatocytes  <cit> .
neither the overexpression of arf <dig> variants nor the absence of arf <dig> resulted in a detectable peroxisomal phenotype.
together, these observations suggest that peroxisome-bound arf <dig> alone is not essential for peroxisome biogenesis in mammalian cells.
the lack of a phenotypic defect is most probably the result of functional redundancy among individual arf proteins  <cit> , and since it has been reported that exogenously added arf <dig> can bind to isolated rat liver peroxisomes  <cit> , we decided to carry out another series of experiments in which arf6wt, arf6t27n, or arf6q67l were transiently co-overexpressed with either wild-type arf <dig> or a mutant defective in gdp-binding  or gtp-hydrolysis .
these experiments showed that a combined overexpression of arf <dig> and arf <dig> variants resulted in a  mislocalization of newly-synthesized peroxisomal reporter proteins and an altered peroxisome morphology.
importantly, the intracellular transport pathway of hslk2-myc-his, a human mitochondrial multisubstrate lipid kinase  <cit> , was not disturbed.
interestingly, combinations with arf1t31n and arf6t27n consistently displayed the strongest phenotypes.
similar observations were observed in cho and hepg <dig> cells .
these mutants are thought to render endogenous arf <dig> and arf <dig> inactive, presumably by sequestering a select set of guanine nucleotide exchange factors  <cit> .
since a similar, but less drastic, phenotype was observed when other arf <dig> and arf <dig> variants were expressed, and arf <dig>  arf <dig>  and arf <dig> could not substitute for arf <dig>  it is unlikely that the  mislocalization of the peroxisomal reporter proteins is the result of indirect changes in the level of activation of other arf subtypes.
in addition, our observation that a similar phenotype was obtained with all arf <dig> and arf <dig> variants also suggests the necessity for a completion of the gdp/gtp cycle of these proteins to achieve their potential function in peroxisome biogenesis .
in summary, these findings – which suggest that arf <dig> and arf <dig> act in tandem to regulate a peroxisomal trafficking pathway – lend strong support to and extend the hypothesis that the cooperation of two arf proteins at the same subcellular location is a general feature of arf signaling  <cit> .
to date, only one group has studied the potential relationship between adp-ribosylation factors and peroxisome formation in mammalian cells, and shown that exogenously added arf <dig> and arf <dig> can bind to isolated rat liver peroxisomes  <cit> .
in this work, we have extended these findings by showing that  also endogenous arf <dig> associates with the cytoplasmic side of the peroxisomal membrane, and  a combined overexpression of arf <dig> and arf <dig> variants selectively impairs peroxisomal protein import and alters peroxisome morphology.
the molecular mechanisms underlying these phenotypic alterations remain to be clarified, since these arf proteins do not interact directly with any of the currently identified human peroxins as tested with yeast- and bacterial two-hybrid assays .
nevertheless, based on  a recent report showing that active arf <dig> can recruit arno – a soluble arf <dig> gtpase-activating guanine nucleotide exchange factor – to the plasma membrane  <cit> ,  a growing body of evidence supporting the existence of vesicular trafficking pathways between peroxisomes and other subcellular compartments  <cit> , and  the observation that arf <dig> is involved in the transport pathway of tomato bushy stunt virus  <dig> kda replication protein from peroxisomes to the er in nicotiana tabacum cells  <cit> , it is tempting to speculate that activated peroxisomal arf <dig> is involved in the formation of peroxisome-derived vesicles by serving as an adaptor for the recruitment of not yet identified guanine nucleotide exchange factor to sites at the peroxisomal membrane which in turn may lead to arf <dig> activation and cytosolic coat protein recruitment.
depending on the functional redundancy among the arf proteins and the role of the peroxisome-derived vesicles in different organisms, the inability of a cell to form such vesicles may result in an increase in peroxisome number or a block in the formation of new organelles.
CONCLUSIONS
in this study, we provide evidence that endogenous arf <dig>  the mammalian orthologue of s. cerevisiae arf <dig>  can tightly associate with the outside face of the peroxisomal membrane.
we also demonstrated that a combined overexpression of arf <dig> and arf <dig> variants caused mislocalization of newly-synthesized peroxisomal proteins and resulted in an alteration of peroxisome morphology.
these observations suggest that arf <dig> – albeit not essential – is a key player in mammalian peroxisome biogenesis.
in addition, these findings extend the concept that specific arf isoform pairs may act in tandem to regulate exclusive trafficking pathways.
the precise mechanism by which arf <dig> functions in peroxisome biogenesis remains to be determined.

 peroxisomes are a diverse group of organelles which accommodate many activities related to lipid metabolism. in mammals, these organelles harbor a number of essential metabolic functions including ether phospholipid biosynthesis, fatty acid α – and β-oxidation, and glyoxylate detoxification. the size, number and enzyme content of peroxisomes can be affected by various environmental, metabolic, and developmental factors. peroxisomes are thought to arise de novo from the endoplasmic reticulum or multiply by fission of pre-existing peroxisomes. the relative contribution of both pathways to the total number of peroxisomes in wild-type cells is not yet clear, and the molecular mechanisms governing these processes are only beginning to be unraveled. studies in different model systems have demonstrated that the pex11p family of peroxisomal membrane proteins, a select set of dynamin-related proteins, and fis and mff – two putative adaptors for drps – coordinately control the shape, size and number of peroxisomes in a cell. the pex proteins are supposed to be involved in the elongation and tubulation of pre-existing peroxisomes, whereas the drps, fis and mff are thought to catalyze the final fission event. it has been put forward also that adp-ribosylation factors and coatomer proteins are involved in the biosynthetic process of the peroxisomal membrane. arguments in favor of this hypothesis are the observations that arf arf and copi coatomer proteins can bind to highly purified rat liver peroxisomes, expression of a temperature-sensitive ε-cop subunit in cho cells at non-permissive temperature exhibited a dramatic change in peroxisome morphology, and the s. cerevisiae orthologues of mammalian arf and arf regulate peroxisome division up and down, respectively. also, peroxisomal arf/copi might be involved in the retrieval of cargo from peroxisomes back to the er. the observation that a dominant-negative mutant of arf inhibits the transport pathway of tomato bushy stunt virus kda replication protein from peroxisomes to the er in nicotiana tabacum cells supports this hypothesis. arf and arf are – together with sar – the most comprehensively studied proteins of the adp-ribosylation factor family. members of this group of small gtpases are believed to control vesicular trafficking and organelle structure by recruiting coat proteins, to function as regulators of phospholipid metabolism, and to modulate cytoskeletal organization. arf has been shown to function in the gtp-dependent recruitment of copi coatomer to budding transport vesicles that move from the golgi to the er. in addition, it has been shown that arf1-gtp regulates the recruitment of clathrin to late golgi and endosome compartments through binding with heterotetrameric adaptor protein complexes as well as monomeric golgi-localized γ-ear-containing arf-binding proteins. arf a protein predominantly associated with the plasma membrane, is thought to regulate endosomal membrane delivery and traffic, membrane raft trafficking, membrane recycling during phagocytosis, and actin remodeling at the cell periphery. in this study, we dissect the potential role of small gtpases, and especially of arf in peroxisome biogenesis.  yeast cells deficient in arf or arf exhibit a pronounced increase in peroxisome number on oleate compared to wild-type cells in view of a continuously increasing number of studies reporting that the er may provide peroxisomes with lipids and some membrane proteins in the form of a preperoxisomal structure, we investigated whether or not yeast strains deficient in one of the small gtpases that function as a molecular switch in protein trafficking contained functional peroxisomes. as such, all viable null strains of the rab and the sar1/arf families were tested for their ability to consume oleate as a sole carbon source, a growth condition requiring functional peroxisomes. a wild-type strain and a strain lacking pex5p – the import receptor for peroxisomal matrix proteins containing a c-terminal peroxisomal targeting signal – were included as appropriate controls, and oleate consumption was scored by halo formation. none of the yeast strains deficient in one of the small gtpases displayed significant changes in halo formation compared to the wild-type strain. this observation indicates that the corresponding gtpases are not essential for peroxisome biogenesis in yeast. this conclusion is in line with our observations that none of these strains displayed a detectable peroxisomal sorting defect for the peroxisomal matrix protein marker egfp-pts or the peroxisomal membrane protein marker pex11p-egfp. interestingly, the average number of peroxisomes in oleate-grown cells was substantially increased in the Δarf and Δarf strains compared to the wild-type strain. to determine whether or not these changes were significant, the values obtained for the wild-type and arf-null strains were analyzed more thoroughly. a one-way anova showed significant differences among the groups. a subanalysis by the tukey-kramer multiple comparisons procedure revealed that the peroxisome number in oleate-grown Δarf and Δarf yeast cells did indeed significantly differ from that in oleate-grown wild-type cells. by contrast, there was no significant pairwise difference between the peroxisome numbers in oleate-grown Δarf and wild-type cells. summarized, these observations indicate that, at least under certain growth conditions, arf and arf may be involved in the regulation of peroxisome number in s. cerevisiae. interestingly, the construction and analysis of a double Δarf1Δarf deletion mutant revealed a cumulative effect of each individual deletion when the cells were grown on oleate. copurification of arf proteins with rat liver peroxisomes in order to investigate whether or not any endogenous arf proteins are associated with mammalian peroxisomes, we probed rat liver subcellular fractions with 1d a mouse monoclonal antibody that binds to a linear epitope found in arf proteins but not in other gtp-binding proteins. these experiments revealed the presence of a weak but detectable kda 1d9-immunoreactive band in the purified peroxisomal fraction. by performing membrane floatation experiments and employing arf-specific antibodies, we could show that this protein was membrane-associated and most likely arf. consistent with this identification is the observation that antibodies towards arf arf arf and/or arf but not arf did not yield a signal above background. note that the anti-arf antibody employed in this study does not cross-react with arf arf or arf, and is only yielding a detectable signal for arf when this protein is expressed in the high-nanogram to low-microgram range. arf was not included in these experiments, as this protein – which is absent in humans – is 96% identical to arf. the results obtained with the anti-arf antibody were not conclusive. that is, a very weak signal could be observed, but only after very long exposure times. however, as the epitope recognized by the monoclonal antibody directed against arf is unknown, we can not exclude the possibility of some cross-reactivity with arf finally, as subcellular fractionation cannot exclude with absolute certainty that a particular protein is located in a contaminating organelle that co-purifies with the organelle under study, and the peroxisomal fractions employed also contained small amounts of glutamate dehydrogenase, bip/grp, and pan-cadherin, we subjected peroxisomal fractions with different degrees of purity to immunoelectron microscopy. these experiments established that the anti-arf 1d and anti-arf antibodies specifically recognized a protein on the outer aspect of the peroxisomal membrane, and not on the membrane of a contaminating organelle. arf is not essential for peroxisome biogenesis in mouse hepatocytes in order to investigate the potential role of arf in peroxisome biogenesis, we examined the number of peroxisomes, peroxisomal morphology, and the localization of peroxisomal membrane and matrix proteins in arf6-/- fetal hepatocytes. as it is well-known that peroxisomes in cultured hepatoma cells may exhibit distinct alterations of shape, size, and distribution dependent on culture conditions, we analyzed high, middle and low density hepatocyte populations from wild-type and knockout animals side-by-side. probing these cells with antibodies raised against the peroxisomal membrane protein pex14p, the pts protein catalase, the peroxisomal membrane protein pmp or the pts protein thiolase showed that ablation of arf did not visibly alter the localization of peroxisomal proteins in cultured fetal hepatocytes from both control and clofibrate-treated pregnant mice. clofibrate is a hypolipidemic drug known to induce peroxisome proliferation in rodent liver. in addition, no evidence could be found for an altered number or spatial distribution of peroxisomes. these findings may be surprising in light of our observations that the number of peroxisomes is altered in s. cerevisiae cells deficient in arf, the yeast orthologue of mammalian arf. we suggest that a functional redundancy of different arf proteins may protect peroxisome biogenesis in mammals. in this context it is interesting to point out that every combination of double – but not single – knockdowns of arf arf arf and arf yielded a distinct pattern of defects in secretory and endocytic traffic in hela cells. co-overexpression of arf and arf variants impairs peroxisomal protein import in mammalian cells as a previous study has shown that exogenously added arf can bind to highly purified rat liver peroxisomes, we investigated whether or not overexpression of wild-type, gtp hydrolysis-defective or gtp binding-defective variants of arf and arf, individually or in pairwise combinations, affected peroxisomal protein import in mammalian cells. these experiments demonstrated that overexpression of individual arf variants had no effect on the localization of egfp-pts a peroxisomal matrix protein reporter. however, when arf and arf variants were simultaneously expressed, all combinations yielded a mislocalization of the reporter protein to various degrees. interestingly, co-expression of the gdp-bound dominant-negative mutants arf6t27n and arf1t31n resulted in a partial to complete cytosolic localization of egfp-pts in approximately 80% of the cells ). similar findings were observed for hspmp34-myc-his, a pmp reporter protein. this observation indicates that the mislocalization of egfp-pts is most likely the indirect result of a peroxisomal membrane protein import deficiency. at first sight, this hypothesis seems to be at odds with the observation that a simultaneous expression of arf6t27n and arf1t31n did not have a visible effect on the subcellular localization of endogenous pex14p. however, as the half-life of peroxisomes in cultured mammalian cells is approximately two days, and the turnover rate of pex14p is rather low, the pex14p signals observed in figure 7b most likely represent pre-existing pex14p molecules which were already localized to peroxisomes before arf6t27n and arf1t31n were co-overexpressed. interestingly, upon closer inspection, we found that the staining pattern of pex14p was slightly changed in at least some cells co-overexpressing arf6t27n and arf1t31n. indeed, a diffuse'background' staining could be observed, and – upon a drastic enlargement of the images – it became also clear that the appearance of the pex14p-immunoreactive particles was affected. the precise nature and mechanism of these changes remain to be determined. importantly, arf arf and arf could not substitute for arf in these assays. in addition, co-expression of the gdp-bound dominant-negative variants of arf and arf did not interfere with the sorting of hslk2-myc-his, a mitochondrial reporter protein. discussion in many instances, the formation of membrane-delimited compartments from pre-existing organelles is regulated by small gtpases of the rab and arf family. a growing amount of evidence suggests that monomeric gtp-binding proteins may also play a role in peroxisome biogenesis. for example, mass spectrometry-based quantitative proteomic profiling studies have shown that in yeast rho is recruited to peroxisomes upon their induction by oleate, and it has been suggested that this protein regulates the assembly state of actin on the peroxisome membrane, thereby controlling peroxisome membrane dynamics and biogenesis. recently, it has also been shown that mya an isoform of arabidopsis thaliana myosin xi, is targeted to peroxisomes through an interaction with atrab2ca. furthermore, mammalian arf and arf have been shown to bind to isolated rat liver peroxisomes, and scarf and scarf the yeast orthologues of these proteins, have been implicated in the control of peroxisome proliferation. a similar function has been suggested for trypanosoma brucei arf a protein which shares characteristics with both arf and arf and has a vital role in the maintenance of the endocytic pathway. interestingly, no arf proteins have been identified in proteome studies of yeast and mammalian peroxisomes. a possible explanation might be that the relative abundance of these proteins in the analyzed samples is extremely low. in this study, we initially investigated whether or not yeast strains deficient in arf arf arf arl cin vps ypt ypt ypt ypt ypt ypt ypt or ypt were capable of growing on oleate as a sole carbon source and/or displayed a peroxisomal protein sorting defect. the results of these experiments demonstrated that all these strains, which embody every viable yeast deletion mutant of the rab and sar1/arf subfamilies of small gtpases, contain functional peroxisomes. interestingly, the average number of peroxisomes in oleate-grown cells was significantly upregulated in the Δarf and Δarf strains compared to the wild-type strain, and this phenotype was even more pronounced in the double Δarf1Δarf deletion mutant. this observation indicates that scarf and scarf dampen peroxisome proliferation, either directly or indirectly. although this result confirms previous observations that scarf and scarf are implicated in the control of peroxisome proliferation, it is not consistent with the authors' conclusion that scarf is required for the oleate-induced peroxisome proliferation in s. cerevisiae. the reasons for the discrepancy between our results and the results previously presented might be several. for example, while we were making use of an arf deletion mutant, lay et al. utilized an arf temperature-sensitive mutant in an Δarf background. also, while these authors analyzed the peroxisome number per cell after hours of growth on oleate, we analyzed the cells after or days of growth on oleate. note that after transfer of the cells to oleate-containing medium, the number of peroxisomes per cell continuously increased as a function of time for at least days, in the Δarf and Δarf strains compared to the wild-type strain. at this point, it is not clear how arf and arf suppress oleate-induced peroxisome proliferation in s. cerevisiae. one possibility might be that these proteins are involved in the generation of peroxisome-derived vesicles, which transport er-derived factors from peroxisomes back to the er or deliver metabolites, lipids, or proteins to mitochondria. disturbances in such pathways would result in an increase in peroxisome number. we next investigated whether or not endogenous arf proteins were associated with the membrane of rat liver peroxisomes. in a first series of experiments, we obtained evidence that 1d a mouse monoclonal antibody with specificity for mammalian arf arf arf arf arf and – to a lesser extent – arf, recognized a kda band in highly purified peroxisomal membrane preparations. we could show that this kda protein was associated with the outside face of the peroxisomal membrane, and identify this protein as arf. this protein, which is considered to be the mammalian orthologue of yeast arf has been reported to act in a wide range of processes, including endocytosis, cytokinesis, phagocytosis, and the organization of the actin cytoskeleton. our observation that endogenous arf is also associated with peroxisomes may seem surprising given that the subcellular localization of this protein has already been documented in several reports. however, as peroxisomes occupy less than 2% of the cell volume, a partial association of arf with these organelles may have been easily missed. also, as the strength of the signal and its detection depend heavily on the amount of protein and may be easily masked by stronger signals, the methods employed might not be sensitive enough. no evidence could be obtained for the association of any other endogenous arf protein with the peroxisomal membrane. however, these results should be considered with caution as such proteins may dissociate during the purification procedure and be present on the peroxisomal membrane in concentrations below the detection limits of the employed arf-specific antibodies. to study the function of arf in peroxisome biogenesis, we first examined the number of peroxisomes, peroxisomal morphology, and the location of peroxisomal membrane and matrix proteins in ptk cells transiently overexpressing either wild-type arf or a mutant defective in gtp-binding or gtp-hydrolysis and arf6-/- fetal mouse hepatocytes. neither the overexpression of arf variants nor the absence of arf resulted in a detectable peroxisomal phenotype. together, these observations suggest that peroxisome-bound arf alone is not essential for peroxisome biogenesis in mammalian cells. the lack of a phenotypic defect is most probably the result of functional redundancy among individual arf proteins, and since it has been reported that exogenously added arf can bind to isolated rat liver peroxisomes, we decided to carry out another series of experiments in which arf6wt, arf6t27n, or arf6q67l were transiently co-overexpressed with either wild-type arf or a mutant defective in gdp-binding or gtp-hydrolysis. these experiments showed that a combined overexpression of arf and arf variants resulted in a mislocalization of newly-synthesized peroxisomal reporter proteins and an altered peroxisome morphology. importantly, the intracellular transport pathway of hslk2-myc-his, a human mitochondrial multisubstrate lipid kinase, was not disturbed. interestingly, combinations with arf1t31n and arf6t27n consistently displayed the strongest phenotypes. similar observations were observed in cho and hepg cells. these mutants are thought to render endogenous arf and arf inactive, presumably by sequestering a select set of guanine nucleotide exchange factors. since a similar, but less drastic, phenotype was observed when other arf and arf variants were expressed, and arf arf and arf could not substitute for arf it is unlikely that the mislocalization of the peroxisomal reporter proteins is the result of indirect changes in the level of activation of other arf subtypes. in addition, our observation that a similar phenotype was obtained with all arf and arf variants also suggests the necessity for a completion of the gdp/gtp cycle of these proteins to achieve their potential function in peroxisome biogenesis. in summary, these findings – which suggest that arf and arf act in tandem to regulate a peroxisomal trafficking pathway – lend strong support to and extend the hypothesis that the cooperation of two arf proteins at the same subcellular location is a general feature of arf signaling. to date, only one group has studied the potential relationship between adp-ribosylation factors and peroxisome formation in mammalian cells, and shown that exogenously added arf and arf can bind to isolated rat liver peroxisomes. in this work, we have extended these findings by showing that also endogenous arf associates with the cytoplasmic side of the peroxisomal membrane, and a combined overexpression of arf and arf variants selectively impairs peroxisomal protein import and alters peroxisome morphology. the molecular mechanisms underlying these phenotypic alterations remain to be clarified, since these arf proteins do not interact directly with any of the currently identified human peroxins as tested with yeast- and bacterial two-hybrid assays. nevertheless, based on a recent report showing that active arf can recruit arno – a soluble arf gtpase-activating guanine nucleotide exchange factor – to the plasma membrane, a growing body of evidence supporting the existence of vesicular trafficking pathways between peroxisomes and other subcellular compartments, and the observation that arf is involved in the transport pathway of tomato bushy stunt virus kda replication protein from peroxisomes to the er in nicotiana tabacum cells, it is tempting to speculate that activated peroxisomal arf is involved in the formation of peroxisome-derived vesicles by serving as an adaptor for the recruitment of not yet identified guanine nucleotide exchange factor to sites at the peroxisomal membrane which in turn may lead to arf activation and cytosolic coat protein recruitment. depending on the functional redundancy among the arf proteins and the role of the peroxisome-derived vesicles in different organisms, the inability of a cell to form such vesicles may result in an increase in peroxisome number or a block in the formation of new organelles.  in this study, we provide evidence that endogenous arf the mammalian orthologue of s. cerevisiae arf can tightly associate with the outside face of the peroxisomal membrane. we also demonstrated that a combined overexpression of arf and arf variants caused mislocalization of newly-synthesized peroxisomal proteins and resulted in an alteration of peroxisome morphology. these observations suggest that arf – albeit not essential – is a key player in mammalian peroxisome biogenesis. in addition, these findings extend the concept that specific arf isoform pairs may act in tandem to regulate exclusive trafficking pathways. the precise mechanism by which arf functions in peroxisome biogenesis remains to be determined. 
these observations suggest that arf is a key player in mammalian peroxisome biogenesis. however, co-overexpression of wild-type, gtp hydrolysis-defective or gtp binding-defective forms of arf and arf caused mislocalization of newly-synthesized peroxisomal proteins and resulted in an alteration of peroxisome morphology. in addition, they also lend strong support to and extend the concept that specific arf isoform pairs may act in tandem to regulate exclusive trafficking pathways. here we show that all viable saccharomyces cerevisiae strains deficient in one of the small gtpases which have an important role in the regulation of vesicular transport contain functional peroxisomes, and that the number of these organelles in oleate-grown cells is significantly upregulated in the arf and arf null strains compared to the wild-type strain. despite this, ablation of arf did neither influence the regulation of peroxisome abundance nor affect the localization of peroxisomal proteins in cultured fetal hepatocytes. it has been suggested that adp-ribosylation factors and copi coatomer complexes are involved in these processes. in addition, we provide evidence that a portion of endogenous arf the mammalian orthologue of yeast arf is associated with the cytoplasmic face of rat liver peroxisomes.
 peroxisomes execute diverse and vital functions in virtually every eukaryote. new peroxisomes form by budding from pre-existing organelles or de novo by vesiculation of the er. it has been suggested that adp-ribosylation factors and copi coatomer complexes are involved in these processes.  here we show that all viable saccharomyces cerevisiae strains deficient in one of the small gtpases which have an important role in the regulation of vesicular transport contain functional peroxisomes, and that the number of these organelles in oleate-grown cells is significantly upregulated in the arf and arf null strains compared to the wild-type strain. in addition, we provide evidence that a portion of endogenous arf the mammalian orthologue of yeast arf is associated with the cytoplasmic face of rat liver peroxisomes. despite this, ablation of arf did neither influence the regulation of peroxisome abundance nor affect the localization of peroxisomal proteins in cultured fetal hepatocytes. however, co-overexpression of wild-type, gtp hydrolysis-defective or gtp binding-defective forms of arf and arf caused mislocalization of newly-synthesized peroxisomal proteins and resulted in an alteration of peroxisome morphology.  these observations suggest that arf is a key player in mammalian peroxisome biogenesis. in addition, they also lend strong support to and extend the concept that specific arf isoform pairs may act in tandem to regulate exclusive trafficking pathways. 