Arjen M. Krikken

Dynamin-related proteins Vps1p and Dnm1p control peroxisome abundance in Saccharomyces cerevisiae

Saccharomyces cerevisiae contains three dynamin-related-proteins, Vps1p, Dnm1p and Mgm1p. Previous data from glucose-grown VPS1 and DNM1 null mutants suggested that Vps1p, but not Dnm1p, plays a role in regulating peroxisome abundance. Here we show that deletion of DNM1 also results in reduction of peroxisome numbers. This was not observed in glucose-grown dnm1 cells, but was evident in cells grown in the presence of oleate. Similar observations were made in cells lacking Fis1p, a protein involved in Dnm1p function. Fluorescence microscopy of cells producing Dnm1-GFP or GFP-Fis1p demonstrated that both proteins had a dual localization on mitochondria and peroxisomes. Quantitative analysis revealed a greater reduction in peroxisome number in oleate-induced vps1 cells relative to dnm1 or fis1 cells. A significant fraction of oleate-induced vps1 cells still contained two or more peroxisomes. Conversely, almost all cells of a dnm1 vps1 double-deletion strain contained only one, enlarged peroxisome. This suggests that deletion of DNM1 reinforces the vps1 peroxisome phenotype. Time-lapse imaging indicated that during budding of dnm1 vps1 cells, the single peroxisome present in the mother cell formed long protrusions into the developing bud. This organelle divided at a very late stage of the budding process, possibly during cytokinesis.

Transcriptional down-regulation of peroxisome numbers affects selective peroxisome degradation in Hansenula polymorpha.

We have isolated and characterized a novel transcription factor of Hansenula polymorpha that is involved in the regulation of peroxisomal protein levels. This protein, designated Mpp1p, belongs to the family of Zn(II)2Cys6 proteins. In cells deleted for the function of Mpp1p the levels of various proteins involved in peroxisome biogenesis (peroxins) and function (enzymes) are reduced compared with wild type or, in the case of the matrix protein dihydroxyacetone synthase, fully absent. Also, upon induction of mpp1 cells on methanol, the number of peroxisomes was strongly reduced relative to wild type cells and generally amounted to one organelle per cell. Remarkably, this single organelle was not susceptible to selective peroxisome degradation (pexophagy) and remained unaffected during exposure of methanol-induced cells to excess glucose conditions. We show that this mechanism is a general phenomenon in H. polymorpha in the case of cells that contain only a single peroxisome.

Preperoxisomal vesicles can form in the absence of Pex3

Contrary to earlier findings, preperoxisomal membrane structures form in yeast cells lacking the peroxin Pex3 and are competent to mature into functional peroxisomes upon Pex3 reintroduction.

Peroxisome fission in Hansenula polymorpha requires Mdv1 and Fis1, two proteins also involved in mitochondrial fission

We show that Mdv1 and Caf4, two components of the mitochondrial fission machinery in Saccharomyces cerevisiae, also function in peroxisome proliferation. Deletion of MDV1, CAF4 or both, however, had only a minor effect on peroxisome numbers at peroxisome‐inducing growth conditions, most likely related to the fact that Vps1 – and not Dnm1 – is the key player in peroxisome fission in this organism. In contrast, in Hansenula polymorpha, which has only a Dnm1‐dependent peroxisome fission machinery, deletion of MDV1 led to a drastic reduction of peroxisome numbers. This phenotype was accompanied by a strong defect in mitochondrial fission. The MDV1 paralog CAF4 is absent in H. polymorpha. In wild‐type H. polymorpha, cells Dnm1–mCherry and green fluorescent protein (GFP)–Mdv1 colocalize in spots that associate with both peroxisomes and mitochondria. Furthermore, Fis1 is essential to recruit Mdv1 to the peroxisomal and mitochondrial membrane. However, formation of GFP–Mdv1 spots – and related to this normal organelle fission – is strictly dependent on the presence of Dnm1. In dnm1 cells, GFP–Mdv1 is dispersed over the surface of peroxisomes and mitochondria. Also, in H. polymorpha mdv1 or fis1 cells, the number of Dnm1–GFP spots is strongly reduced. These spots still associate to organelles but are functionally inactive.

The Hansenula polymorpha ATG25 gene encodes a novel coiled-coil protein that is required for macropexophagy

We have isolated the Hansenula polymorpha ATG11 and ATG25 genes, which are both required for glucose-induced selective peroxisome degradation (macropexophagy). ATG11 was identified before in other yeast species and shown to be involved in the Cvt pathway in Saccharomyces cerevisiae and glucose-induced micropexophagy in Pichia pastoris. Our data indicate that HpATG11 is required for macropexophagy. ATG25 represents a novel gene that encodes a 45 kDa coiled-coil protein. We show that this protein co-localizes with Atg11 on a small structure, which most likely represents the pre-autophagosomal structure (PAS). Cells of a constructed ATG25 deletion strain (atg25) displayed relatively slow, continuous degradation of peroxisomes by microautophagy during growth on methanol in the presence of excess nitrogen that also continued after induction of selective peroxisome degradation. This suggests that the processes of selective and non-selective autophagy are dysregulated in atg25 cells.

Peroxisome reintroduction in Hansenula polymorpha requires Pex25 and Rho1

De novo peroxisome formation in peroxisome-deficient yeast cells requires Rho1 and the Pex11 family protein Pex25.

Hansenula polymorpha pex11 cells are affected in peroxisome retention

We have cloned and characterized the Hansenula polymorpha PEX11 gene. Our morphological data are consistent with previous observations that peroxisome proliferation can be regulated by modulating Pex11p levels. Surprisingly, pex11 cells also showed a defect in peroxisome retention in mother cells during vegetative cell reproduction. Until now, Saccharomyces cerevisiae Inp1p has been the only peroxisomal protein that has been shown to play a role in the organelle retention process. H. polymorpha inp1 cells are also affected in peroxisome retention, like pex11 cells. We show by time‐lapse imaging that Inp1–green fluorescent protein localization varies during the cell cycle and that the protein is normally recruited to peroxisomes in pex11 cells. Taken together, our data show that H. polymorpha Pex11p is not only important for peroxisome proliferation but is also required for proper peroxisome segregation during cell division.

Atg8 is essential for macropexophagy in Hansenula polymorpha

We have isolated a peroxisome‐degradation‐deficient (pdd) mutant of the methylotrophic yeast Hansenula polymorpha via gene tagging mutagenesis. Sequencing revealed that the mutant was affected in the HpATG8 gene. HpAtg8 is a protein with high sequence similarity to both Pichia pastoris and Saccharomyces cerevisiae Atg8 and appeared to be essential for selective peroxisome degradation (macropexophagy) and nitrogen‐limitation induced microautophagy. Fluorescence microscopy revealed that a GFP.Atg8 fusion protein was located close to the vacuole. After induction of macropexophagy, the GFP.Atg8 containing spot extended to engulf an individual peroxisome. In cells of a constructed deletion strain, sequestration of individual organelles was never completed; analysis of series of serial sections revealed that invariably a minor diaphragm‐like opening remained. We hypothesize that H. polymorpha Atg8 facilitates sealing of the sequestering membranes during selective peroxisome degradation.

Degeneration of penicillin production in ethanol-limited chemostat cultivations of Penicillium chrysogenum: A systems biology approach

BackgroundIn microbial production of non-catabolic products such as antibiotics a loss of production capacity upon long-term cultivation (for example chemostat), a phenomenon called strain degeneration, is often observed. In this study a systems biology approach, monitoring changes from gene to produced flux, was used to study degeneration of penicillin production in a high producing Penicillium chrysogenum strain during prolonged ethanol-limited chemostat cultivations.ResultsDuring these cultivations, the biomass specific penicillin production rate decreased more than 10-fold in less than 22 generations. No evidence was obtained for a decrease of the copy number of the penicillin gene cluster, nor a significant down regulation of the expression of the penicillin biosynthesis genes. However, a strong down regulation of the biosynthesis pathway of cysteine, one of the precursors of penicillin, was observed. Furthermore the protein levels of the penicillin pathway enzymes L-α-(δ-aminoadipyl)-L-α-cystenyl-D-α-valine synthetase (ACVS) and isopenicillin-N synthase (IPNS), decreased significantly. Re-cultivation of fully degenerated cells in unlimited batch culture and subsequent C-limited chemostats did only result in a slight recovery of penicillin production.ConclusionsOur findings indicate that the observed degeneration is attributed to a significant decrease of the levels of the first two enzymes of the penicillin biosynthesis pathway, ACVS and IPNS. This decrease is not caused by genetic instability of the penicillin amplicon, neither by down regulation of the penicillin biosynthesis pathway. Furthermore no indications were obtained for degradation of these enzymes as a result of autophagy. Possible causes for the decreased enzyme levels could be a decrease of the translation efficiency of ACVS and IPNS during degeneration, or the presence of a culture variant impaired in the biosynthesis of functional proteins of these enzymes, which outcompeted the high producing part of the population.

The membrane remodeling protein Pex11p activates the GTPase Dnm1p during peroxisomal fission.

Significance Peroxisomal fission is crucial for cell viability because peroxisome fission defects cause severe disease. The initial step in peroxisomal fission, membrane elongation, requires the membrane remodeling protein Peroxin 11 (Pex11p). Here, we identify an additional function for Pex11p, demonstrating that Pex11p also plays a crucial role in the final step of peroxisomal fission: membrane separation. We show that Pex11p functions as a GTPase activating protein (GAP) for Dynamin-related 1 (Dnm1p) and that this GAP activity is conserved from yeast to mammalians. This work identifies a previously unknown requirement for a GAP in dynamin-like protein function. The initial phase of peroxisomal fission requires the peroxisomal membrane protein Peroxin 11 (Pex11p), which remodels the membrane, resulting in organelle elongation. Here, we identify an additional function for Pex11p, demonstrating that Pex11p also plays a crucial role in the final step of peroxisomal fission: dynamin-like protein (DLP)-mediated membrane scission. First, we demonstrate that yeast Pex11p is necessary for the function of the GTPase Dynamin-related 1 (Dnm1p) in vivo. In addition, our data indicate that Pex11p physically interacts with Dnm1p and that inhibiting this interaction compromises peroxisomal fission. Finally, we demonstrate that Pex11p functions as a GTPase activating protein (GAP) for Dnm1p in vitro. Similar observations were made for mammalian Pex11β and the corresponding DLP Drp1, indicating that DLP activation by Pex11p is conserved. Our work identifies a previously unknown requirement for a GAP in DLP function.