Functional Complementation

Abstract

Many studies on gene cloning, particularly on genes with a very low level of expression, have benefited from the so‐called functional cloning of genes, mostly complementary DNAs (deoxyribonucleic acids) in higher eukaryotes including mammals, by phenotype‐complementation assay, using cell mutants deficient in biological pathways. Successful gene‐cloning studies using a rapid functional complementation assay of mammalian somatic cell mutants include the search for pathogenic genes responsible for peroxisome biogenesis disorders (PBDs), autosomal recessive, progressive disorders characterised by loss of multiple peroxisomal metabolic functions and defects in peroxisome assembly, consisting of 14 complementation groups (CGs). Such a forward genetic approach using a dozen CGs of peroxisome‐deficient Chinese hamster ovary (CHO) cell mutants led to isolation of human peroxin (PEX) genes. Search for pathogenic genes responsible for PBDs of all 14 CGs is now accomplished with another approach, the homology search by screening the human expressed sequence tag (EST) database using yeast PEX genes.

Key concepts

  • Mutation is a permanent change in the DNA sequence of a gene. Mutations in a gene's DNA sequence can alter the amino acid sequence of the protein encoded by the gene.
  • Mutations may lead to changes in phenotype. Cell mutants are a highly useful tool in genetic, biochemical and cell biological research.
  • Eukaryotic organisms have two primary cell types – germ and somatic. Mutations can occur in either cell type. If a gene is altered in a germ cell, the mutation is termed a germinal mutation. Because germ cells give rise to gametes, some gametes will carry the mutation and it will be passed on to the next generation when the individual successfully mates.
  • Somatic mutations are genetic alterations acquired by a cell that can be passed to the progeny of the mutated cell in the course of cell division. Somatic mutations differ from germ line mutations, which are inherited genetic alterations that occur in the germ cells (i.e. sperm and eggs). Somatic mutations are frequently caused by environmental factors, such as exposure to ultraviolet radiation or to certain chemicals.
  • In genetics, complementation occurs when two strains of an organism with different homozygous recessive mutations that produce the same phenotype (e.g. a change in wing structure in flies) produce offspring with the wild‐type phenotype when mated or crossed. Complementation will occur only if the mutations are in different genes.
  • A genetic disorder is an illness caused by one or more abnormalities in the genome, especially a condition that is present from birth (congenital). Most genetic disorders are quite rare and affect one person in every several thousands or millions.
  • Several methods including the most recent lipofection have been developed for transfecting DNA into animal cells.
  • Genetic phenotype‐complementation of peroxisome assembly‐defective mutants of mammalian somatic cells such as Chinese hamster ovary (CHO) cells and of several yeast species including Saccharomyces cerevisiae and Pichia pastoris would lead to identification and characterisation of numerous genes that are essential for peroxisome biogenesis.

Keywords: cell mutants; phenotype; complementation; peroxisome biogenesis disorders; PEX genes

Figure 1. Schematic view of peroxisome biogenesis in mammals. The intracellular locations and molecular properties of peroxins are shown. Peroxins are divided into three groups: (1) peroxins that are required for matrix protein import; (2) those including Pex3p, Pex16p and Pex19p, responsible for peroxisome membrane assembly; (3) those such as three forms of Pex11p, Pex11pα, Pex11pβ and Pex11pγ, apparently involved in peroxisome proliferation where DLP1, Mff and Fis1 coordinately function. PTS1 and PTS2 proteins are recognised by Pex5p and Pex7p, respectively, in the cytoplasm. Two isoforms, Pex5pS and Pex5pL, of Pex5p are identified in mammals. PTS1 proteins are transported by homo‐ and hetero‐oligomers of Pex5pS and Pex5pL to peroxisomes, where Pex14p functions as a convergent, initial docking site of the ‘protein import machinery’ translocon. Pex5pL directly interacts with the PTS2 receptor, Pex7p, carrying its cargo PTS2 protein in the cytosol and translocates the Pex7p–PTS2 protein complex to Pex14p. PTS1 and PTS2 proteins are then released at the inner surface and/or inside of peroxisomes, downstream Pex14p and upstream Pex13p. Pex5p and Pex7p subsequently translocate to other translocon components such as the RING peroxins, Pex2p, Pex10p and Pex12p. Both Pex5p and Pex7p finally shuttle back to the cytosol. In regard to peroxisome‐cytoplasmic shuttling of Pex5p, Pex5p initially targets to an 800‐kDa complex containing Pex14p and then translocates to a 500‐kDa complex comprising RING peroxins. At the terminal step of the protein import reaction, Pex1p and Pex6p of the AAA family catalyse the export of Pex5p, where ubiquitination of Pex5p is a prerequisite for the Pex5p exit. A cytosolic factor, AWP1/ZFAND6 (p40), is required for the export and recycling of Ub‐Pex5p in mammals.
close

References

Barøy T, Koster J, Strømme P, et al. (2015) A novel type of rhizomelic chondrodysplasia punctata, RCDP5, is caused by loss of the PEX5 long isoform. Human Molecular Genetics 24: 5845–5854.

Ebberink MS, Koster J, Visser G, et al. (2012) A novel defect of peroxisome division due to a homozygous non‐sense mutation in the PEX11β gene. Journal of Medical Genetics 49: 307–313.

Fujiki Y (2000) Peroxisome biogenesis and peroxisome biogenesis disorders. FEBS Letters 476: 42–46.

Fujiki Y, Okumoto K, Kinoshita N and Ghaedi K (2006) Lessons from peroxisome‐deficient Chinese hamster ovary (CHO) cell mutants. Biochimica et Biophysica Acta‐Molecular Cell Research 1763: 1374–1381.

Fujiki Y, Yagita Y and Matsuzaki T (2012) Peroxisome biogenesis disorders: molecular basis for impaired peroxisomal membrane assembly‐ In metabolic functions and biogenesis of peroxisomes in health and disease. Biochimica et Biophysica Acta‐Molecular Basis of Disease 1822: 1337–1342.

Fujiki Y, Okumoto K, Mukai S, Honsho M and Tamura S (2014) Peroxisome biogenesis in mammalian cells. Frontiers in Physiology 5: article 307.

Gould SJ, Raymond GV and Valle D (2001) The peroxisome biogenesis disorders. In: Scriver CR, Beaudet AI, Sly WS and Valle D (eds) The Metabolic Basis of Inherited Disease, 8th edn, pp. 3181–3217. New York: McGraw‐Hill.

Hosoi K, Miyata N, Mukai S, et al. (2017) The VDAC2‐BAK axis regulates peroxisomal membrane permeability. Journal of Cell Biology 216: 709–721.

Islinger M, Grille S, Fahimi HD and Schrader M (2012) The peroxisome: an update on mysteries. Histochemistry and Cell Biology 137: 547–574.

de Laat WL, Jaspers NG and Hoeijmakers JH (1999) Molecular mechanism of nucleotide excision repair. Genes and Development 13: 768–785.

Lazarow PB and Fujiki Y (1985) Biogenesis of peroxisomes. Annual Review of Cell Biology 1: 489–530.

Lazarow PB and Moser HW (1995) Disorders of peroxisome biogenesis. In: Scriver CR, Beaudet AI, Sly WS and Valle D (eds) The Metabolic Basis of Inherited Disease, pp. 2287–2324. New York: McGraw‐Hill.

Matsumoto N, Tamura S and Fujiki Y (2003a) The pathogenic peroxin Pex26p recruits the Pex1p‐Pex6p AAA ATPase complexes to peroxisomes. Nature Cell Biology 5: 454–460.

Matsumoto N, Tamura S, Furuki S, et al. (2003b) Mutations in novel peroxin gene PEX26 that cause peroxisome biogenesis disorders of complementation group 8 provide a genotype‐phenotype correlation. American Journal of Human Genetics 73: 233–246.

Morand OH, Allen L‐AH, Zoeller RA and Raetz CRH (1990) A rapid selection for animal cell mutants with defective peroxisomes. Biochimica et Biophysica Acta 1034: 132–141.

Okumoto K, Bogaki A, Tateishi K, et al. (1997) Isolation and characterization of peroxisome‐deficient Chinese hamster ovary cell mutants representing human complementation group III. Experimental Cell Research 233: 11–20.

Sambrook J and Russell DW (2001) Molecular Cloning: A Laboratory Manual, 3rd edn, vol. 2, pp. 11.67–11.78. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.

Schatz G and Dobberstein B (1996) Common principles of protein translocation across membranes. Science 271: 1519–1526.

Schliebs W, Girzalsky W and Erdmann R (2010) Peroxisomal protein import and ERAD: variations on a common theme. Nature Review. Molecular Cell Biology 11: 885–890.

Shimozawa N, Tsukamoto T, Suzuki Y, et al. (1992a) A human gene responsible for Zellweger syndrome that affects peroxisome assembly. Science 255: 1132–1134.

Shimozawa N, Tsukamoto T, Suzuki Y, et al. (1992b) Animal cell mutants represent two complementation groups of peroxisome‐defective Zellweger syndrome. Journal of Clinical Investigation 90: 1864–1870.

Subramani S, Koller A and Snyder WB (2000) Import of peroxisomal matrix and membrane proteins. Annual Review of Biochemistry 69: 399–418.

Tateishi K, Okumoto K, Shimozawa N, et al. (1997) Newly identified Chinese hamster ovary cell mutants defective in peroxisome biogenesis represent two novel complementation groups in mammals. European Journal of Cell Biology 73: 352–359.

Tsukamoto T, Yokota S and Fujiki Y (1990) Isolation and characterization of Chinese hamster ovary cell mutants defective in assembly of peroxisomes. Journal of Cell Biology 110: 651–660.

Tsukamoto T, Miura S and Fujiki Y (1991) Restoration by a 35K membrane protein of peroxisome assembly in a peroxisome‐deficient mammalian cell mutant. Nature 350: 77–81.

Wanders RJA and Waterham HR (2006) Biochemistry of mammalian peroxisomes revisited. Annual Review of Biochemistry 75: 295–332.

Weller S, Gould SJ and Valle D (2003) Peroxisome biogenesis disorders. Annual Review of Genomics and Human Genetics 4: 165–211.

Wickner W and Schekman R (2005) Protein translocation across biological membranes. Science 310: 1452–1456.

Zoeller RA and Raetz CRH (1986) Isolation of animal cell mutants deficient in plasmalogen biosynthesis and peroxisome assembly. Proceedings of the National Academy of Sciences of the United States of America 83: 5170–5174.

Further Reading

Alberts B, Johnson A, Lewis J, et al. (eds) (2008) Manipulating proteins, DNA, and RNA. In: Molecular Biology of the Cell, 5th edn, pp. 501–578. New York, NY: Garland Science.

Baes M, Gressens P, Baumgart E, et al. (1997) A mouse model for Zellweger syndrome. Nature Genetics 17: 49–57.

Faust PL and Hatten ME (1997) Targeted deletion of the PEX2 peroxisome assembly gene in mice provides a model for Zellweger syndrome, a human neuronal migration disorder. Journal of Cell Biology 139: 1293–1305.

Maxwell M, Bjorkman J, Nguyen T, et al. (2003) Pex13 inactivation in the mouse disrupts peroxisome biogenesis and leads to a Zellweger syndrome phenotype. Molecular and Cellular Biology 23: 5947–5957.

Web Links

Peroxisome biogenesis disorders; PeroxisomeDB: www.peroxisomedb.org/

OMIM database in NCBI; OMIM Entry # 214100 (Zellweger syndrome) https://www.omim.org/entry/214100

Contact Editor close
Submit a note to the editor about this article by filling in the form below.

* Required Field

How to Cite close
Fujiki, Yukio(Sep 2017) Functional Complementation. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1002/9780470015902.a0005676.pub3]