The peroxisomal membrane targeting elements of human peroxin 2 (PEX2).

Peroxin 2 (PEX2) is a 35-kDa integral peroxisomal membrane protein with two transmembrane regions and a zinc RING domain within its cytoplasmically exposed C-terminus. Although its role in peroxisome biogenesis and function is poorly understood, it seems to be involved in peroxisomal matrix protein import. PEX2 is synthesized on free cytosolic ribosomes and is posttranslationally imported into the peroxisome membrane by specific targeting information. While a clear picture of the basic targeting mechanisms for peroxisomal matrix proteins has emerged over the past years, the targeting processes for peroxisomal membrane proteins are less well understood. We expressed various deletion constructs of PEX2 in fusion with the green fluorescent protein in COS-7 cells and determined their intracellular localization. We found that the minimum peroxisomal targeting signal of human PEX2 consists of an internal protein region of 30 amino acids (AA130 to AA159) and the first transmembrane domain, and that adding the second transmembrane domain increases targeting efficiency. Within the minimum targeting region we identified the motif "KX6(I/L)X(L/F/I)LK(L/F/I)" that includes important targeting information and is also present in the targeting regions of the 22-kDa peroxisomal membrane protein (PMP22) and the 70-kDa peroxisomal membrane protein (PMP70). Mutations in this targeting motif mislocalize PEX2 to the cytosol. In contrast, the second transmembrane domain does not seem to have specific peroxisomal membrane targeting information. Replacing the second transmembrane domain of human PEX2 with the transmembrane domain of human cytochrome c oxidase subunit IV does not alter PEX2 peroxisome targeting function and efficiency.

Relationship between expression of KAI1 metastasis suppressor gene, mRNA levels and p53 in human bladder and prostate cancer cell lines.

The molecular basis for the loss of KAI1 expression in invasive and metastatic tumors and tumor cell lines is not understood. Recently, identification of a sequence with homology to the consensus p53-binding motif in the promoter of the KAI1 metastasis suppressor gene, has led to a proposal that transcriptional regulation by p53 controls expression of KAI1, and that a dramatic down-regulation of KAI1 mRNA levels in invasive tumors and many tumor cell lines, is directly due to loss of p53 function. We have tested this hypothesis by assessing KAI1 mRNA levels in a series of 22 cell lines derived from bladder and prostate cancers, in which we confirmed the p53 gene sequence and characterized the functional status of the endogenous p53 protein. We anticipated that cell lines expressing p53 capable of transactivation should express high levels of KAI1 mRNA compared with cell lines expressing defective p53, or which were p53-null. KAI1 mRNA levels were determined by northern analysis using a full-length KAI1 cDNA probe, and varied widely between cell lines examined. However, there was no association between these levels and p53 status. Furthermore, transfection of representative cell lines with wild-type p53, or exposure to DNA damaging agents, had no effect on KAI1 mRNA levels. Our data suggest that p53 is not a major factor regulating levels of KAI1 mRNA in bladder and prostate cancer cell lines.

PEX1 mutations in complementation group 1 of Zellweger spectrum patients correlate with severity of disease.

The peroxisome biogenesis disorders (PBD) are a group of autosomal-recessive diseases with complex developmental and metabolic phenotypes, including the Zellweger spectrum and rhizomelic chondrodysplasia punctata. The diseases are caused by defects in peroxisomal matrix protein import and are characterized by the loss of multiple peroxisomal metabolic functions. In humans, 12 complementation groups have been identified, with complementation group 1 accounting for more than two thirds of all PBD patients. Mutations in the PEX1 gene encoding a member of the AAA protein family of ATPases are responsible for the defects in this group, and a variety of PEX1 mutant alleles have been described. We characterized the PEX1 gene mutations and associated haplotypes in a group of thoroughly documented Zellweger spectrum patients in complementation group 1 who represent the broad range of phenotypic variation. We compared the type of mutation with the age of survival, clinical manifestations, and biochemical alterations and found a close relationship between genotype and age of survival. Missense mutations cause a milder form of disease, whereas insertions, deletions, and nonsense mutations are associated with severe clinical phenotypes. Thus, knowing the PEX1 gene mutation is helpful in predicting the course of disease in individual cases.

Two different targeting signals direct human peroxisomal membrane protein 22 to peroxisomes.

The 22-kDa peroxisomal membrane protein (PMP22) is a major component of peroxisomal membranes in mammals. Although its precise role in peroxisome function is poorly understood, it seems to be involved in pore forming activity and may contribute to the unspecific permeability of the organelle membrane. PMP22 is synthesized on free cytosolic ribosomes and then directed to the peroxisome membrane by specific targeting information. Previous studies in rats revealed that PMP22 contains one distinct peroxisomal membrane targeting signal in the amino-terminal cytoplasmic tail. We cloned and characterized the targeting signal of human PMP22 and compared it with the already described characteristics of the corresponding rat protein. Amino acid sequence alignment of rat and human protein revealed 77% identity including a high conservation of several protein motifs. We expressed various deletion constructs of PMP22 in fusion with the green fluorescent protein in COS-7 cells and determined their intracellular localization. In contrast to previous studies on rat PMP22 and most other peroxisomal membrane proteins, we showed that human as well as rat PMP22 contains two distinct and nonoverlapping peroxisomal membrane targeting signals, one in the amino-terminal and the other in the carboxyl-terminal protein region. They consist of two transmembrane domains and adjacent protein loops with almost identical basic clusters. Both of these peroxisomal targeting regions interact with PEX19, a factor required for peroxisome membrane synthesis. In addition, we observed that fusing the green fluorescent protein immediately adjacent to the targeting region completely abolishes targeting function and mislocalizes PMP22 to the cytosol.