Proteins that carry dual targeting signals can act as tethers between peroxisomes and partner organelles.

Peroxisomes are organelles with crucial functions in oxidative metabolism. To correctly target to peroxisomes, proteins require specialized targeting signals. A mystery in the field is the sorting of proteins that carry a targeting signal for peroxisomes and as well as for other organelles, such as mitochondria or the endoplasmic reticulum (ER). Exploring several of these proteins in fungal model systems, we observed that they can act as tethers bridging organelles together to create contact sites. We show that in Saccharomyces cerevisiae this mode of tethering involves the peroxisome import machinery, the ER-mitochondria encounter structure (ERMES) at mitochondria and the guided entry of tail-anchored proteins (GET) pathway at the ER. Our findings introduce a previously unexplored concept of how dual affinity proteins can regulate organelle attachment and communication.

Sharing the wealth: The versatility of proteins targeted to peroxisomes and other organelles.

Peroxisomes are eukaryotic organelles with critical functions in cellular energy and lipid metabolism. Depending on the organism, cell type, and developmental stage, they are involved in numerous other metabolic and regulatory pathways. Many peroxisomal functions require factors also relevant to other cellular compartments. Here, we review proteins shared by peroxisomes and at least one different site within the cell. We discuss the mechanisms to achieve dual targeting, their regulation, and functional consequences. Characterization of dual targeting is fundamental to understand how peroxisomes are integrated into the metabolic and regulatory circuits of eukaryotic cells.

Engineering Ustilago maydis for production of tailor-made mannosylerythritol lipids.

Mannosylerythritol lipids (MELs) are surface active glycolipids secreted by various fungi. MELs can be used as biosurfactants and are a biodegradable resource for the production of detergents or pharmaceuticals. Different fungal species synthesize a unique mixture of MELs differing in acetyl- and acyl-groups attached to the sugar moiety. Here, we report the construction of a toolbox for production of glycolipids with predictable fatty acid side chains in the basidiomycete Ustilago maydis. Genes coding for acyl-transferases involved in MEL production (Mac1 and Mac2) from different fungal species were combined to obtain altered MEL variants with distinct physical properties and altered antimicrobial activity. We also demonstrate that a U. maydis paralog of the acyltransferase Mac2 with a different substrate specificity can be employed for the biosynthesis of modified MEL variants. In summary, our data showcase how the fungal repertoire of Mac enzymes can be used to engineer tailor-made MELs according to specific biotechnological or pharmaceutical requirements.

Peroxisomal targeting of a protein phosphatase type 2C via mitochondrial transit.

Correct intracellular distribution of proteins is critical for the function of eukaryotic cells. Certain proteins are targeted to more than one cellular compartment, e.g. to mitochondria and peroxisomes. The protein phosphatase Ptc5 from Saccharomyces cerevisiae contains an N-terminal mitochondrial presequence followed by a transmembrane domain, and has been detected in the mitochondrial intermembrane space. Here we show mitochondrial transit of Ptc5 to peroxisomes. Translocation of Ptc5 to peroxisomes depended both on the C-terminal peroxisomal targeting signal (PTS1) and N-terminal cleavage by the mitochondrial inner membrane peptidase complex. Indirect targeting of Ptc5 to peroxisomes prevented deleterious effects of its phosphatase activity in the cytosol. Sorting of Ptc5 involves simultaneous interaction with import machineries of both organelles. We identify additional mitochondrial proteins with PTS1, which localize in both organelles and can increase their physical association. Thus, a tug-of-war-like mechanism can influence the interaction and communication of two cellular compartments.

Non-AUG Translation Initiation Generates Peroxisomal Isoforms of 6-Phosphogluconate Dehydrogenase in Fungi.

Proteins destined for transport to specific organelles usually contain targeting information, which are embedded in their sequence. Many enzymes are required in more than one cellular compartment and different molecular mechanisms are used to achieve dual localization. Here we report a cryptic type 2 peroxisomal targeting signal encoded in the 5' untranslated region of fungal genes coding for 6-phosphogluconate dehydrogenase (PGD), a key enzyme of the oxidative pentose phosphate pathway. The conservation of the cryptic PTS2 motif suggests a biological function. We observed that translation from a non-AUG start codon generates an N-terminally extended peroxisomal isoform of Ustilago maydis PGD. Non-canonical initiation occurred at the sequence AGG AUU, consisting of two near-cognate start codons in tandem. Taken together, our data reveal non-AUG translation initiation as an additional mechanism to achieve the dual localization of a protein required both in the cytosol and the peroxisomes.

The Obvious and the Hidden: Prediction and Function of Fungal Peroxisomal Matrix Proteins.

Fungal peroxisomes are characterized by a number of specific biological functions. To understand the physiology and biochemistry of these organelles knowledge of the proteome content is crucial. Here, we address different strategies to predict peroxisomal proteins by bioinformatics approaches. These tools range from simple text searches to network based learning strategies. A complication of this analysis is the existence of cryptic peroxisomal proteins, which are overlooked in conventional bioinformatics queries. These include proteins where targeting information results from transcriptional and posttranscriptional alterations. But also proteins with low efficiency targeting motifs that are predominantly localized in the cytosol, and proteins lacking any canonical targeting information, can play important roles within peroxisomes. Many of these proteins are so far unpredictable. Detection and characterization of these cryptic peroxisomal proteins revealed the presence of novel peroxisomal enzymatic reaction networks in fungi.

Ribosomal readthrough at a short UGA stop codon context triggers dual localization of metabolic enzymes in Fungi and animals.

Translation of mRNA into a polypeptide chain is a highly accurate process. Many prokaryotic and eukaryotic viruses, however, use leaky termination of translation to optimize their coding capacity. Although growing evidence indicates the occurrence of ribosomal readthrough also in higher organisms, a biological function for the resulting extended proteins has been elucidated only in very few cases. Here, we report that in human cells programmed stop codon readthrough is used to generate peroxisomal isoforms of cytosolic enzymes. We could show for NAD-dependent lactate dehydrogenase B (LDHB) and NAD-dependent malate dehydrogenase 1 (MDH1) that translational readthrough results in C-terminally extended protein variants containing a peroxisomal targeting signal 1 (PTS1). Efficient readthrough occurs at a short sequence motif consisting of a UGA termination codon followed by the dinucleotide CU. Leaky termination at this stop codon context was observed in fungi and mammals. Comparative genome analysis allowed us to identify further readthrough-derived peroxisomal isoforms of metabolic enzymes in diverse model organisms. Overall, our study highlights that a defined stop codon context can trigger efficient ribosomal readthrough to generate dually targeted protein isoforms. We speculate that beyond peroxisomal targeting stop codon readthrough may have also other important biological functions, which remain to be elucidated.

Fungal peroxisomes as biosynthetic organelles.

Peroxisomes are nearly ubiquitous single-membrane organelles harboring multiple metabolic pathways beside their prominent role in the ß-oxidation of fatty acids. Here we review the diverse metabolic functions of peroxisomes in fungi. A variety of fungal metabolites are at least partially synthesized inside peroxisomes. These include the essential co-factor biotin but also different types of secondary metabolites. Peroxisomal metabolites are often derived from acyl-CoA esters for example ß-oxidation intermediates. In several ascomycetes a subtype of peroxisomes has been identified that is metabolically inactive but is required to plug the septal pores of wounded hyphae. Thus, peroxisomes are versatile organelles that can adapt their function to the life style of an organism. This remarkable variability suggests that the full extent of the biosynthetic capacity of peroxisomes is still elusive. Moreover, in fungi peroxisomes are non-essential under laboratory conditions making them attractive organelles for biotechnological approaches and the design of novel metabolic pathways in customized peroxisomes.

Peroxisomes contribute to biosynthesis of extracellular glycolipids in fungi.

Many microorganisms secrete surface-active glycolipids. The basidiomycetous fungus Ustilago maydis produces two different classes of glycolipids, mannosylerythritol lipids (MEL) and ustilagic acids (UAs). Here we report that biosynthesis of MELs is partially localized in peroxisomes and coupled to peroxisomal fatty acid degradation. The acyltransferases, Mac1 and Mac2, which acylate mannosylerythritol with fatty acids of different length, contain a type 1 peroxisomal targeting signal (PTS1). We demonstrate that Mac1 and Mac2 are targeted to peroxisomes, while other enzymes involved in MEL production reside in different compartments. Mis-targeting of Mac1 and Mac2 to the cytosol did not block MEL synthesis but promoted production of MEL species with altered acylation pattern. This is in contrast to peroxisome deficient mutants that produced MELs similar to the wild type. We could show that cytosolic targeting of Mac1 and Mac2 reduces the amount of UA presumably due to competition for overlapping substrates. Interestingly, hydroxylated fatty acids characteristic for UAs appear in MELs corroborating cross-talk between both biosynthesis pathways. Therefore, peroxisomal localization of MEL biosynthesis is not only prerequisite for generation of the natural spectrum of MELs, but also facilitates simultaneous assembly of different glycolipids in a single cell.