A recent model for peroxisome biogenesis postulates that peroxisomes form de novo continuously in wild-type cells by heterotypic fusion of endoplasmic reticulum–derived vesicles containing distinct sets of peroxisomal membrane proteins. This model proposes a role in vesicle fusion for the Pex1/Pex6 complex, which has an established role in matrix protein import. The growth and division model proposes that peroxisomes derive from existing peroxisomes. We tested these models by reexamining the role of Pex1/Pex6 and dynamin-related proteins in peroxisome biogenesis. We found that induced depletion of Pex1 blocks the import of matrix proteins but does not affect membrane protein delivery to peroxisomes; markers for the previously reported distinct vesicles colocalize in pex1 and pex6 cells; peroxisomes undergo continued growth if fission is blocked. Our data are compatible with the established primary role of the Pex1/Pex6 complex in matrix protein import and show that peroxisomes in Saccharomyces cerevisiae multiply mainly by growth and division.

In Saccharomyces cerevisiae, peroxisomes are the sole site of fatty acid β-oxidation. During this process, NAD⁺ is reduced to NADH. When cells are grown on oleate medium, peroxisomal NADH is reoxidised to NAD⁺ by malate dehydrogenase (Mdh3p) and reduction equivalents are transferred to the cytosol by the malate/oxaloacetate shuttle. The ultimate step in lysine biosynthesis, the NAD⁺-dependent dehydrogenation of saccharopine to lysine, is another NAD⁺-dependent reaction performed inside peroxisomes. We have found that in glucose grown cells, both the malate/oxaloacetate shuttle and a glycerol-3-phosphate dehydrogenase 1(Gpd1p)-dependent shuttle are able to maintain the intraperoxisomal redox balance. Single mutants in MDH3 or GPD1 grow on lysine-deficient medium, but an mdh3/gpd1Δ double mutant accumulates saccharopine and displays lysine bradytrophy. Lysine biosynthesis is restored when saccharopine dehydrogenase is mislocalised to the cytosol in mdh3/gpd1Δ cells. We conclude that the availability of intraperoxisomal NAD⁺ required for saccharopine dehydrogenase activity can be sustained by both shuttles. The extent to which each of these shuttles contributes to the intraperoxisomal redox balance may depend on the growth medium. We propose that the presence of multiple peroxisomal redox shuttles allows eukaryotic cells to maintain the peroxisomal redox status under different metabolic conditions.

The architecture of the cytoskeleton and its remodeling are tightly regulated by dynamic reorganization of keratin-rich intermediate filaments. Plakin family proteins associate with the network of intermediate filaments (IFs) and affect its reorganization during migration, differentiation, and response to stress. The smallest plakin, periplakin (PPL), interacts specifically with intermediate filament proteins K8, K18, and vimentin via its C-terminal linker domain. Here, we show that periplakin is SUMOylated at a conserved lysine in its linker domain (K1646) preferentially by SUMO1. Our data indicate that PPL SUMOylation is essential for the proper reorganization of the keratin IF network. Stresses perturbing intermediate-filament and cytoskeletal architecture induce hyper-SUMOylation of periplakin. Okadaic acid induced hyper-phosphorylation dependent collapse of the keratin IF network results in a similar hyper-SUMOylation of PPL. Strikingly, exogenous over-expression of a non-SUMOylatable periplakin mutant (K1646R) induced aberrant bundling and loose network interconnections of the keratin filaments. Time-lapse imaging of cells expressing the K1646R mutant showed the enhanced sensitivity of keratin filament collapse upon okadaic acid treatment. Our data identify an important regulatory role for periplakin SUMOylation in dynamic reorganization and stability of keratin IFs. Movie S1: Movie of GFP-PPL-C transfected HeLa cells, recorded for 3.5hr at 100X. Download Original Video (2.9 MB) https://ascb-prod-streaming.literatumonline.com/journals/content/mboc/0/mboc.ahead-of-print/mbc.e18-04-0244/20181203/media/mc-e18-04-0244-s01.,1200,900,600,300,150,.mp4.m3u8?b92b4ad1b4f274c70877518410abb28bf45a6fadfff54c0884ea58110075615c1d5919ea753725730a4097154a8f56c8491b2e70b0e87b5e9f35b4d61387d2130c8df350edbabb355e8ef985f87fe75bc1a1492f8a9620d0da1a66aa4ead290f14b5b16eb2d82ff7bdfc1f72a709d1207b010ba8631a89255ce0c445d37c3546643da9e22aafcf2a72cdeff53a04a31a688e8fe129ed07617585164f828c249b403c497e9187eaa5164719d09e30 Movie S2: Movie of GFP-PPL-C K1646R transfected HeLa cells, recorded for 3.5hr 100X. Download Original Video (4.3 MB) https://ascb-prod-streaming.literatumonline.com/journals/content/mboc/0/mboc.ahead-of-print/mbc.e18-04-0244/20181203/media/mc-e18-04-0244-s02.,1200,900,600,300,150,.mp4.m3u8?b92b4ad1b4f274c70877518410abb28bf45a6fadfff54c0884ea58110075615c1d5919ea753725730a4097154a8f56c8491b2e70b0e87b5e9f35b4d61387d2130c8df350edbabb355e8ef985f87fe75bc1a1492f8a9620d0da1a66aa4ead290f14b5b16eb2d82ff7bdfc1f72a709d1207b010ba8631a89255fe0c445d37c3546844b6ceb8449eae3840180098d461f189050d89a8f092f871aa0321d49c10126e223c32259d5e5692aca1273daa5 Movie S3: Movie of GFP-PPL-C transfected HeLa cells, recorded for 3.5hr in the presence of 0.5μM OA. Download Original Video (1.7 MB) https://ascb-prod-streaming.literatumonline.com/journals/content/mboc/0/mboc.ahead-of-print/mbc.e18-04-0244/20181203/media/mc-e18-04-0244-s03.,1200,900,600,300,150,.mp4.m3u8?b92b4ad1b4f274c70877518410abb28bf45a6fadfff54c0884ea58110075615c1d5919ea753725730a4097154a8f56c8491b2e70b0e87b5e9f35b4d61387d2130c8df350edbabb355e8ef985f87fe75bc1a1492f8a9620d0da1a66aa4ead290f14b5b16eb2d82ff7bdfc1f72a709d1207b010ba8631a89255ee0c445d37c35460a827583be2d2553e43cabd0d73795a3b3d460c59354d92ae830974a7cec514026778b88d7b1fc4dc99ee45beb0b Movie S4: Movie of GFP-PPL-C K1646R transfected HeLa cells, recorded for 3.5hr in the presence of 0.5μM OA. Download Original Video (2.2 MB) https://ascb-prod-streaming.literatumonline.com/journals/content/mboc/0/mboc.ahead-of-print/mbc.e18-04-0244/20181203/media/mc-e18-04-0244-s04.,1200,900,600,300,150,.mp4.m3u8?b92b4ad1b4f274c70877518410abb28bf45a6fadfff54c0884ea58110075615c1d5919ea753725730a4097154a8f56c8491b2e70b0e87b5e9f35b4d61387d2130c8df350edbabb355e8ef985f87fe75bc1a1492f8a9620d0da1a66aa4ead290f14b5b16eb2d82ff7bdfc1f72a709d1207b010ba8631a892559e0c445d37c3546d0f2c1ab32d1fa28d36299aa5817d67e28b350cf5886984b5cbbc90ad78e689f9b7676cd8f62cfc94db40babc57e Movie S5: Movie of GFP-PPL-C transfected HeLa cells, recorded for 3.5hr at 100X in the presence of 0.25μM OA. Download Original Video (1.7 MB) https://ascb-prod-streaming.literatumonline.com/journals/content/mboc/0/mboc.ahead-of-print/mbc.e18-04-0244/20181203/media/mc-e18-04-0244-s05.,1200,900,600,300,150,.mp4.m3u8?b92b4ad1b4f274c70877518410abb28bf45a6fadfff54c0884ea58110075615c1d5919ea753725730a4097154a8f56c8491b2e70b0e87b5e9f35b4d61387d2130c8df350edbabb355e8ef985f87fe75bc1a1492f8a9620d0da1a66aa4ead290f14b5b16eb2d82ff7bdfc1f72a709d1207b010ba8631a892558e0c445d37c3546a2d18d4a01d948953f93bf4190e6540bf688b33280fe46338c5d30bf9ee1ae5456fde96adae91e878623d1408ca5 Movie S6: Movie of GFP-PPL-C K1646R transfected HeLa cells, recorded for 3.5hr at 100X in the presence of 0.25μM OA. Download Original Video (1.8 MB) https://ascb-prod-streaming.literatumonline.com/journals/content/mboc/0/mboc.ahead-of-print/mbc.e18-04-0244/20181203/media/mc-e18-04-0244-s06.,1200,900,600,300,150,.mp4.m3u8?b92b4ad1b4f274c70877518410abb28bf45a6fadfff54c0884ea58110075615c1d5919ea753725730a4097154a8f56c8491b2e70b0e87b5e9f35b4d61387d2130c8df350edbabb355e8ef985f87fe75bc1a1492f8a9620d0da1a66aa4ead290f14b5b16eb2d82ff7bdfc1f72a709d1207b010ba8631a89255be0c445d37c354637da21ac9ca130afad5549cd7862d66cd661d973935bffb2c09a9f28468c6797333a1740ce41a0e5119083b5e7f8 Movie S7: Movie of GFP-PPL-C transfected HEK293T cells, recorded for 2hr in the presence of 0.5μM OA. Download Original Video (.7 MB) https://ascb-prod-streaming.literatumonline.com/journals/content/mboc/0/mboc.ahead-of-print/mbc.e18-04-0244/20181203/media/mc-e18-04-0244-s07.,900,600,300,150,.mp4.m3u8?b92b4ad1b4f274c7087751851dabb28b5eff7736ed2d13488ba0685b5d4b43f735f6b48a41f0861e362653b46f872d70e7a199ee5ab054e5562a0a61d56adcc26fec2da2c8c4e10d496af1d512ae0eae5a6a55162ee836212e306de7a32364f84831aa2fda4e2e65549862939fe666834cd0ca5ee206f766002ff2fb7b1d43f603244ec0ab2ca8b9d6ae8e9aeb0bf4b6974435de3735e87d98b08bcf9ab8a30406ff8f5eebf2d760e2 Movie S8: Movie of GFP-PPL-C K1646R transfected HEK293T cells, recorded for 2hr in the presence of 0.5μM OA. Download Original Video (.7 MB) https://ascb-prod-streaming.literatumonline.com/journals/content/mboc/0/mboc.ahead-of-print/mbc.e18-04-0244/20181203/media/mc-e18-04-0244-s08.,900,600,300,150,.mp4.m3u8?b92b4ad1b4f274c7087751851dabb28b5eff7736ed2d13488ba0685b5d4b43f735f6b48a41f0861e362653b46f872d70e7a199ee5ab054e5562a0a61d56adcc26fec2da2c8c4e10d496af1d512ae0eae5a6a55162ee836212e306de7a32364f84831aa2fda4e2e65549862939fe666834cd0ca5ee206f7660f2ff2fb7b1d43f6620b958d5e2dbca577c9a5f0a03dad5dc1bea755bb0214bccf25beddba93d1aabc9aca14797b3e3e87 Movie S9: Movie of GFP-PPL-C transfected MCF7a cells, recorded for 2hr in the presence of 0.5μM OA. Download Original Video (.7 MB) https://ascb-prod-streaming.literatumonline.com/journals/content/mboc/0/mboc.ahead-of-print/mbc.e18-04-0244/20181203/media/mc-e18-04-0244-s09.,900,600,300,150,.mp4.m3u8?b92b4ad1b4f274c7087751851dabb28b5eff7736ed2d13488ba0685b5d4b43f735f6b48a41f0861e362653b46f872d70e7a199ee5ab054e5562a0a61d56adcc26fec2da2c8c4e10d496af1d512ae0eae5a6a55162ee836212e306de7a32364f84831aa2fda4e2e65549862939fe666834cd0ca5ee206f7660e2ff2fb7b1d43f691471eb1f8eb370b94b924777942d1c51500c1724dae32c176bb90dfc9266b8f9e0f49597e7ccb7cf8 Movie S10: Movie of GFP-PPL-C K1646R transfected MCF7a cells, recorded for 2hr in the presence of 0.5μM OA. Download Original Video (.8 MB) https://ascb-prod-streaming.literatumonline.com/journals/content/mboc/0/mboc.ahead-of-print/mbc.e18-04-0244/20181203/media/mc-e18-04-0244-s10.,900,600,300,150,.mp4.m3u8?b92b4ad1b4f274c7087751851dabb28b5eff7736ed2d13488ba0685b5d4b43f735f6b48a41f0861e362653b46f872d70e7a199ee5ab054e5562a0a61d56adcc26fec2da2c8c4e10d496af1d512ae0eae5a6a55162ee836212e306de7a32364f84831aa2fda4e2e65549862939fe666834cd0ca5ee206f767bd7579cc9621db198aec85232b83ab04c4c6a16e01e7d8c027a3556f973a5ca470b8c939f1ed4415c25f8d9d66cb448a82

Dynamin-related proteins (Drps) mediate a variety of membrane remodelling processes. The Saccharomyces cerevisiae Drp, Vps1, is required for endocytosis, endosomal sorting, vacuole fusion, and peroxisome fission and breakdown. How Drps, and in particular Vps1, can function at so many different subcellular locations is of interest to our understanding of cellular organisation. We found that the peroxisomal membrane protein Pex27 is specifically required for Vps1-dependent peroxisome fission in proliferating cells but is not required for Dnm1-dependent peroxisome fission. Pex27 accumulates in constricted regions of peroxisomes and affects peroxisome geometry upon overexpression. Moreover, Pex27 physically interacts with Vps1 in vivo and is required for the accumulation of a GTPase-defective Vps1 mutant (K42A) on peroxisomes. During nitrogen starvation, a condition that halts cell division and induces peroxisome breakdown, Vps1 associates with the pexophagophore. Pex27 is neither required for Vps1 recruitment to the pexophagophore nor for pexophagy. Our study identifies Pex27 as a Vps1-specific partner for the maintenance of peroxisome number in proliferating yeast cells.

Proteins composed of tetratricopeptide repeat (TPR) arrays belong to the α‐solenoid tandem‐repeat family that have unique properties in terms of their overall conformational flexibility and ability to bind to multiple protein ligands. The peroxisomal matrix protein import receptor Pex5 comprises two TPR triplets that recognize protein cargos with a specific C‐terminal Peroxisomal Targeting Signal (PTS) 1 motif. Import of PTS1‐containing protein cargos into peroxisomes through a transient pore is mainly driven by allosteric binding, coupling and release mechanisms, without a need for external energy. A very similar TPR architecture is found in the functionally unrelated TRIP8b, a regulator of the hyperpolarization‐activated cyclic nucleotide‐gated (HCN) ion channel. TRIP8b binds to the HCN ion channel via a C‐terminal sequence motif that is nearly identical to the PTS1 motif of Pex5 receptor cargos. Pex5, Pex5‐related Pex9, and TRIP8b also share a less conserved N‐terminal domain. This domain provides a second protein cargo‐binding site and plays a distinct role in allosteric coupling of initial cargo loading by PTS1 motif‐mediated interactions and different downstream functional readouts. The data reviewed here highlight the overarching role of molecular allostery in driving the diverse functions of TPR array proteins, which could form a model for other α‐solenoid tandem‐repeat proteins involved in translocation processes across membranes.