Reevaluation of the role of Pex1 and dynamin-related proteins in peroxisome membrane biogenesis.

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.

Deficiency of the exportomer components Pex1, Pex6, and Pex15 causes enhanced pexophagy in Saccharomyces cerevisiae.

Turnover of damaged, dysfunctional, or excess organelles is critical to cellular homeostasis. We screened mutants disturbed in peroxisomal protein import, and found that a deficiency in the exportomer subunits Pex1, Pex6, and Pex15 results in enhanced turnover of peroxisomal membrane structures compared with other mutants. Strikingly, almost all peroxisomal membranes were associated with phagophore assembly sites in pex1Δ atg1Δ cells.

Intra-ER sorting of the peroxisomal membrane protein Pex3 relies on its luminal domain.

Pex3 is an evolutionarily conserved type III peroxisomal membrane protein required for peroxisome formation. It is inserted into the ER membrane and sorted via an ER subdomain (the peroxisomal ER, or pER) to peroxisomes. By constructing chimeras between Pex3 and the type III ER membrane protein Sec66, we have been able to separate the signals that mediate insertion of Pex3 into the ER from those that mediate sorting within the ER to the pER subdomain.

Atg36: the Saccharomyces cerevisiae receptor for pexophagy.

Eukaryotic cells adapt their organelle composition and abundance according to environmental conditions. Analysis of the peroxisomal membrane protein Pex3 has revealed that this protein plays a crucial role in peroxisome maintenance as it is required for peroxisome formation, segregation and breakdown. Although its function in peroxisome formation and segregation was known to involve its recruitment to the peroxisomal membrane of factors specific for these processes, the role of Pex3 in peroxisome breakdown was unclear until our recent identification of Atg36 as a novel Saccharomyces cerevisiae Pex3-interacting protein.

Pex3-anchored Atg36 tags peroxisomes for degradation in Saccharomyces cerevisiae.

Peroxisomes undergo rapid, selective autophagic degradation (pexophagy) when the metabolic pathways they contain are no longer required for cellular metabolism. Pex3 is central to the formation of peroxisomes and their segregation because it recruits factors specific for these functions. Here, we describe a novel Saccharomyces cerevisiae protein that interacts with Pex3 at the peroxisomal membrane.

A dual function for Pex3p in peroxisome formation and inheritance.

Saccharomyces cerevisiae Pex3p has been shown to act at the ER during de novo peroxisome formation. However, its steady state is at the peroxisomal membrane, where its role is debated. Here we show that Pex3p has a dual function: one in peroxisome formation and one in peroxisome segregation.

How peroxisomes multiply.

With every cell division, peroxisomes duplicate and are segregated between progeny cells. Here, we discuss the different modes of peroxisome multiplication and the machinery that is involved in each case. Peroxisomes have been considered by many to be peripheral to mainstream cell biology.

Dnm1p-dependent peroxisome fission requires Caf4p, Mdv1p and Fis1p.

Yeast peroxisomes multiply by fission. Fission requires two dynamin-related proteins, Dnm1p and Vps1p. Using an in vivo fission assay, we show that Dnm1p-dependent peroxisome fission requires Fis1p, Caf4p and Mdv1p.

Yeast peroxisomes multiply by growth and division.

Peroxisomes can arise de novo from the endoplasmic reticulum (ER) via a maturation process. Peroxisomes can also multiply by fission. We have investigated how these modes of multiplication contribute to peroxisome numbers in Saccharomyces cerevisiae and the role of the dynamin-related proteins (Drps) in these processes.

HIV-1 Nef-induced down-regulation of MHC class I requires AP-1 and clathrin but not PACS-1 and is impeded by AP-2.

Major histocompatibility complex class I is down-regulated from the surface of human immunodeficiency virus (HIV)-1-infected cells by Nef, a virally encoded protein that is thought to reroute MHC-I to the trans-Golgi network (TGN) in a phosphofurin acidic cluster sorting protein (PACS) 1, adaptor protein (AP)-1, and clathrin-dependent manner. More recently, an alternative model has been proposed, in which Nef uses AP-1 to direct MHC-I to endosomes and lysosomes. Here, we show that knocking down either AP-1 or clathrin with small interfering RNA inhibits the down-regulation of HLA-A2 (an MHC-I isotype) by Nef in HeLa cells.

Functional analysis of AP-2 alpha and mu2 subunits.

The AP-2 adaptor complex plays a key role in cargo recognition and clathrin-coated vesicle formation at the plasma membrane. To investigate the functions of individual binding sites and domains of the AP-2 complex in vivo, we have stably transfected HeLa cells with wild-type and mutant small interfering RNA-resistant alpha and mu2 subunits and then used siRNA knockdowns to deplete the endogenous proteins. Mutating the PtdIns(4,5)P2 binding site of alpha, the phosphorylation site of mu2, or the YXXPhi binding site of mu2 impairs AP-2 function, as assayed by transferrin uptake.

Impaired neuronal migration and endochondral ossification in Pex7 knockout mice: a model for rhizomelic chondrodysplasia punctata.

Rhizomelic chondrodysplasia punctata is a human autosomal recessive disorder characterized by skeletal, eye and brain abnormalities. The disorder is caused by mutations in the PEX7 gene, which encodes the receptor for a class of peroxisomal matrix enzymes. We describe the generation and characterization of a Pex7 mouse knockout (Pex7(-/-)).

Mutational spectrum in the PEX7 gene and functional analysis of mutant alleles in 78 patients with rhizomelic chondrodysplasia punctata type 1.

Rhizomelic chondrodysplasia punctata (RCDP) is a genetically heterogeneous, autosomal recessive disorder of peroxisomal metabolism that is clinically characterized by symmetrical shortening of the proximal long bones, cataracts, periarticular calcifications, multiple joint contractures, and psychomotor retardation. Most patients with RCDP have mutations in the PEX7 gene encoding peroxin 7, the cytosolic PTS2-receptor protein required for targeting a subset of enzymes to peroxisomes. These enzymes are deficient in cells of patients with RCDP, because of their mislocalization to the cytoplasm.