Central role of mic10 in the mitochondrial contact site and cristae organizing system

The mitochondrial contact site and cristae organizing system (MICOS) is a conserved multi-subunit complex crucial for maintaining the characteristic architecture of mitochondria. Studies with deletion mutants identified Mic10 and Mic60 as core subunits of MICOS. Mic60 has been studied in detail; however, topogenesis and function of Mic10 are unknown. We report that targeting of Mic10 to the mitochondrial inner membrane requires a positively charged internal loop, but no cleavable presequence. Both transmembrane segments of Mic10 carry a characteristic four-glycine motif, which has been found in the ring-forming rotor subunit of F1Fo-ATP synthases. Overexpression of Mic10 profoundly alters the architecture of the inner membrane independently of other MICOS components. The four-glycine motifs are dispensable for interaction of Mic10 with other MICOS subunits but are crucial for the formation of large Mic10 oligomers. Our studies identify a unique role of Mic10 oligomers in promoting the formation of inner membrane crista junctions

A Novel Method to Determine the Topology of Peroxisomal Membrane Proteins in Vivo Using the Tobacco Etch Virus Protease

Most proteins essential for the biogenesis of peroxisomes (peroxins) that are identified to date are associated with or are integral components of the peroxisomal membrane. A prerequisite in elucidating their function is to determine their topology in the membrane. We have developed a novel tool to analyze the topology of peroxisomal membrane proteins in the yeast Hansenula polymorpha in vivo using the 27-kDa NIa protease subunit from the tobacco etch virus (TEVp). TEVp specifically cleaves peptides containing the consensus sequence, EXXYXQ↓S (tev). We show that cytosolic TEVp and peroxisomal TEVp.SKL are selectively active on soluble cytosolic and peroxisomal tev-containing proteins in vivo, respectively, without affecting the viability of the yeast cells. The tev sequence was introduced in between the primary sequence of the peroxisomal membrane proteins Pex3p or Pex10p and the reporter protein enhanced green fluorescent protein (eGFP). Co-synthesis of these functional tev-GFP tagged proteins with either cytosolic TEVp or peroxisomal TEVp.SKL revealed that the C termini of Pex3p and Pex10p are exposed to the cytosol. Additional applications of the TEV protease to study peroxisome biogenesis are discussed.

Isolation of mutants of Hansenula polymorpha defective in the assembly of octameric alcohol oxidase

Alcohol oxidase (AO) is a peroxisomal enzyme that catalyses the first step in methanol metabolism in yeast. Monomeric, inactive AO protein is synthesised in the cytosol and subsequently imported into peroxisomes, where the enzymatically active, homo-octameric form is found. The mechanisms involved in AO octamer assembly are largely unclear. Here we describe the isolation of Hansenula polymorpha mutants specifically affected in AO assembly. These mutants are unable to grow on methanol and display reduced AO activities. Based on their phenotypes, three major classes of mutants were isolated. Three additional mutants were isolated that each displayed a unique phenotype. Complementation analysis revealed that the isolated AO assembly mutants belonged to 10 complementation groups.

Glucose-induced and nitrogen-starvation-induced peroxisome degradation are distinct processes in Hansenula polymorpha that involve both common and unique genes

In the methylotrophic yeast Hansenula polymorpha non-selective autophagy, induced by nitrogen starvation, results in the turnover of cytoplasmic components, including peroxisomes. We show that the uptake of these components occurs by invagination of the vacuolar membrane without their prior sequestration and thus differs from the mechanism described for bakers yeast. A selective mode of autophagy in H. polymorpha, namely glucose-induced peroxisome degradation, involves sequestration of individual peroxisomes tagged for degradation by membrane layers that subsequently fuse with the vacuole where the organelle is digested. H. polymorpha pdd mutants are blocked in selective peroxisome degradation. We observed that pdd1-201 is also impaired in non-selective autophagy, whereas this process still normally functions in pdd2-4. These findings suggest that mechanistically distinct processes as selective and non-selective autophagy involve common but also unique genes.

Preperoxisomal vesicles can form in the absence of Pex3

We demonstrate that the peroxin Pex3 is not required for the formation of peroxisomal membrane structures in yeast pex3 mutant cells. Notably, pex3 mutant cells already contain reticular and vesicular structures that harbor key proteins of the peroxisomal receptor docking complex—Pex13 and Pex14—as well as the matrix proteins Pex8 and alcohol oxidase. Other peroxisomal membrane proteins in these cells are unstable and transiently localized to the cytosol (Pex10, Pmp47) or endoplasmic reticulum (Pex11). These reticular and vesicular structures are more abundant in cells of a pex3 atg1 double deletion strain, as the absence of Pex3 may render them susceptible to autophagic degradation, which is blocked in this double mutant. Contrary to earlier suggestions, peroxisomes are not formed de novo from the endoplasmic reticulum when the PEX3 gene is reintroduced in pex3 cells. Instead, we find that reintroduced Pex3 sorts to the preperoxisomal structures in pex3 cells, after which these structures mature into normal peroxisomes.

A Stretch of Positively Charged Amino Acids at the N Terminus of Hansenula polymorpha Pex3p Is Involved in Incorporation of the Protein into the Peroxisomal Membrane

Pex3p is a peroxisomal membrane protein that is essential for peroxisome biogenesis. Here, we show that a conserved stretch of positively charged amino acids (Arg11-X-Lys-Lys-Lys15) in the N terminus of Hansenula polymorpha Pex3p is involved in incorporation of the protein into its target membrane. Despite the strong conservation, this sequence shows a high degree of redundancy. Substitution of either Arg11, Lys13, Lys14, or Lys15 with uncharged or negatively charged amino acids did not interfere with Pex3p location and function. However, a mutant Pex3p, carrying negatively charged amino acids at position 13 and 15 (K13E/K15E), caused moderate but significant defects in peroxisome assembly and matrix protein import. Additional changes in the N terminus of Pex3p, e.g. replacing three or four of the positively charged amino acids with negatively charged ones, led to a typical pex3 phenotype, i.e. accumulation of peroxisomal matrix proteins in the cytosol and absence of peroxisomal remnants. Also, in these cases, the mutant Pex3p levels were reduced. Remarkably, mutant Pex3p proteins were mislocalized to mitochondria or the cytosol, depending on the nature of the mutation. Furthermore, in case of reduced amounts of Pex3p, the levels of other peroxisomal membrane proteins, e.g. Pex10p and Pex14p, were also diminished, suggesting that Pex3p maybe involved in the recruitment or stabilization of these proteins (in the membrane).

Healthcare seeking behaviours among immigrants in Sweden

Bakgrund: Hälsan i sig och möjligheten att söka vård vid nedsatt hälsa ingår i samhällets folkhälsoansvar. Det ansvaret omfattar även de migranter som finns i Sverige. Hur ohälsa tas omhand inom sjukvården och hur hälsoutvecklingen kan stödjas bland migranter har betydelse för hela samhället och samhällsekonomin. Syfte: Att ta reda på mönstret för migranter när det gäller att söka vård, vad man söker för, vilka hinder som kan finnas för vårdsökande, både bland migranterna själva och i vårdapparaten och hur dessa problem kan överbryggas. Metod: Detta är en litteraturbaserad studie. De metoder och den design som användes för att analysera och granska studierna baseras på STROBE och Malteruds granskningsmal för litteraturer studier samt GRADE för metodstyrkan. Resultat: I den systematiska sökningen identifierades 15 artiklar som uppfyllde sökkriterierna. Dessa användes för att svara på frågorna om vårdsökandet och utgjorde underlag för värderingen av den vetenskapliga styrkan när det gäller fynden relaterade till vårdsökandet. Orsaker till vårdsökande omfattade både infektionssjukdomar och icke smittsamma sjukdomar, bland dessa psykisk ohälsa. Det vårdsökande beteendet är bland annat knutet till kulturella uppfattningen om sjukdomars orsaker men också till språkproblem. Sjukvårdens organisation och personalens attityder utgör också hinder för ett adekvat vårdsökande bland migranter. Slutsats: Vårdsökande beteende hos immigranter i Sverige varierar beroende på bakgrund och etnicitet gällande olika sjukdomar och religiös tro. De mönster som påverkar vårdsökande beteendet är dock desamma hos alla grupper och styrs av kulturella och socioekonomiska faktorer liksom av litteracitet och språkbarriärer.Background: Healthcare seeking behaviours among immigrants in Sweden is a public health issue since the health outcome not only affects immigrants but also the Swedish population, the healthcare system and impacts on the national economy. In order to alleviate the problems and support the development of health among immigrants we need a thorough picture of the situation today. Objectives: To explore the healthcare seeking behaviours of immigrants in Sweden looking at the different epidemiological reasons and challenges faced during healthcare seeking and the support than can be appropriate for combating the challenges. Method: Literature based study. The methods and designs applied in the articles were assessed and graded on method strength by STROBE, Malterud guidelines and GRADE respectively. Result: 15 articles were identified through a systematic search and used to determine the quality and strength of the scientific methods used to explore the questions of the study. Reasons for seeking health care were signs and symptoms, communicable as well as non-communicable diseases, among those mental health issues. Obstacles to seeking health care were cultural background as well as perception of causes of diseases and language barriers; in addition, the organisation of care and the attitudes among providers were also seen as obstacles to seeking healthcare. Conclusion: Health seeking behaviour among immigrants vary according to ethnic backgrounds as pertains to diseases and beliefs. However, the challenges faced are common among the different groups and they range from cultural social economic to health illiteracy and language barriers

Razvoj dohodnine v Sloveniji, 1991-2007

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