Biomedical subjects

Arjen M Krikken

Publications and source records attributed to Arjen M Krikken.

8 recordsLinked to original sources

PubMed2006

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.

GTP Phosphohydrolases↗

PubMed2006

Reassembly of peroxisomes in Hansenula polymorpha pex3 cells on reintroduction of Pex3p involves the nuclear envelope.

The reassembly of peroxisomes in Hansenula polymorpha pex3 cells on reintroduction of Pex3p was examined. Using a Pex3-green fluorescent protein (Pex3-GFP) fusion protein, expressed under the control of an inducible promoter, it was observed that, initially on induction of Pex3-GFP synthesis, GFP fluorescence was localized to the endoplasmic reticulum and the nuclear envelope. Subsequently, a single organelle developed per cell that increased in size and multiplied by division. At these stages, GFP fluorescence was confined to peroxisomes. Fractionation experiments on homogenates of pex3 cells, in which the endoplasmic reticulum and nuclear envelope were marked with GFP, identified a small amount of GFP in peroxisomes present in the initial stage of peroxisome reassembly. Our data suggest a crucial role for the endoplasmic reticulum/nuclear envelope in peroxisome reintroduction on complementation of pex3 cells by the PEX3 gene.

Endoplasmic Reticulum↗

PubMed2006

In the yeast Hansenula polymorpha, peroxisome formation from the ER is independent of Pex19p, but involves the function of p24 proteins.

The peroxin Pex19p is important for the formation of functional peroxisomal membranes. Here we show that Hansenula polymorpha Pex19p is also required for peroxisome inheritance. Peroxisome inheritance is partly defective when Pex19p farnesylation is blocked, whereas deletion of PEX19 resulted in a severe defect in partitioning of peroxisomal structures. Time lapse imaging revealed that in newly formed buds, which had not inherited a peroxisome from the mother cell, new peroxisomes are formed that derive from the nuclear envelope/endoplasmic reticulum. This process was impaired upon deletion of EMP24 and ERP3, genes that encode p24 proteins. p24 Proteins are components of coated vesicles that mediate trafficking between the endoplasmic reticulum and Golgi apparatus. In an H. polymorpha wild-type background, deletion of EMP24 and ERP3 resulted in a strong reduction of organelle number in conjunction with an increase in the size of individual peroxisomes. This observation suggests that p24 proteins also play a role in peroxisome development in wild-type H. polymorpha cells.

Endoplasmic Reticulum↗

PubMed2005

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

We have isolated the Hansenula polymorpha ATG25 gene, which is required for glucose-induced selective peroxisome degradation by macropexophagy. ATG25 represents a novel gene that encodes a 45 kDa coiled-coil protein. We show that this protein colocalizes with Atg11 on a small structure, which most likely represents the pre-autophagosomal structure (PAS). In cells of a constructed ATG25 deletion strain (atg25) peroxisomes are constitutively degraded by nonselective microautophagy, a process that in WT H. polymorpha is only observed at nitrogen limitation conditions. This suggests that nonselective microautophagy is deregulated in H. polymorpha atg25 cells.

Autophagy↗

PubMed2005

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.

Amino Acid Sequence↗

PubMed2004

Atg21p is essential for macropexophagy and microautophagy in the yeast Hansenula polymorpha.

ATG genes are required for autophagy-related processes that transport proteins/organelles destined for proteolytic degradation to the vacuole. Here, we describe the identification and characterisation of the Hansenula polymorpha ATG21 gene. Its gene product Hp-Atg21p, fused to eGFP, had a dual location in the cytosol and in peri-vacuolar dots. We demonstrate that Hp-Atg21p is essential for two separate modes of peroxisome degradation, namely glucose-induced macropexophagy and nitrogen limitation-induced microautophagy. In atg21 cells subjected to macropexophagy conditions, sequestration of peroxisomes tagged for degradation is initiated but fails to complete.

Autophagy↗

PubMed2004

Hansenula polymorpha Tup1p is important for peroxisome degradation.

In the yeast Hansenula polymorpha peroxisomes are selectively degraded upon a shift of cells from methanol to glucose-containing media. We identified the H. polymorpha TUP1 gene by functional complementation of the peroxisome degradation deficient mutant pdd2-4. Tup1 proteins function in transcriptional repression of specific sets of genes involved in various cellular processes. Our combined data indicate that H. polymorpha TUP1 is involved in regulation of the switch between peroxisome biogenesis and selective degradation. The initial DNA fragment that complemented H. polymorpha pdd2-4 contained a second gene, encoding H. polymorpha Vps4p. Deletion of the VPS4 gene did not affect selective peroxisome degradation.

Amino Acid Sequence↗

PubMed2003

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.

Aldehyde-Ketone Transferases↗