University of Groningen The Hansenula polymorpha PER8 Gene Encodes a Novel Peroxisomal Integral Membrane Protein Involved in Proliferation

We previously described the isolation of mutants of the methylotrophic yeast Hansenula polymorpha that are defective in peroxisome biogenesis. Here, we describe the characterization of one of these mutants, per8, and the cloning of the PER8 gene. In either methanol or methylamine medium, conditions that normally induce the organdies, per8 cells contain no peroxisome-like structures and peroxisomal enzymes are located in the cytosol. The sequence of PER8 predicts that its product (Per8p) is a novel polypeptide of 34 kD, and antibodies against Per8p recognize a protein of 31 kD. Analysis of the primary sequence of PerSp revealed a 39-amino-acid cysteinerich segment with similarity to the C3HC4 family of zinc-finger motifs. Overexpression of PER8 results in a markedly enhanced increase in peroxisome numbers. We show that PerSp is an integral membrane protein of the peroxisome and that it is concentrated in the membranes of newly formed organdies. We propose that PerSp is a component of the molecular machinery that controls the proliferation of this organelle. UKARYOTIC cells are divided into a variety of membrane-bound compartments or organeUes, each responsible for performing specific metabolic functions. To maintain each organelle, the cell must correctly direct specific sets of proteins to their proper subcellular locations and, as the cell grows and divides, the organelles must be duplicated. For some organdies, such as the endoplasmic reticulnm, mitochondrion, and peroxisome, the cell can also increase organelle numbers in response to certain environmental stimuli (Bolender et al., 1973; Attardi et al., 1988; Braunbeck and V61kl, 1991; Dreyer et al., 1992; Luiken et al., 1992). Of these organelle biogenesis tasks, significant progress has been made in elucidating the molecular mechanisms responsible for protein sorting, whereas little is known regarding the mechanisms responsible for organelle duplication/proliferation. We are interested in understanding biogenesis in peroxisomes (glyoxysomes, glycosomes), a class of single membrane-bound organdies that exists in virtually all cells and is the site of a number of important oxidative reactions (Borst, 1989; Veenhuis and Harder, 1991; van den Bosch, 1992; Subramani, 1993). An unusual characteristic ofperoxisomes relative to other organdies is their functional diverAddress all correspondence to Dr. James M. Cregg, Department of Chemistry, Biochemistry, and Molecular Biology, Oregon Graduate Institute of Science & Technology, P.O. Box 91000, Portland, OR 97291-1000. Ph.: (503) 690-1217. Fax: (503) 6904464. Hans Waterham's present address is Oregon Graduate Institute of Science & Technology, P.O. Box 91000, Portland, OR 97291-1000. sity; that is, the specific metabolic pathways found in the organelle vary depending upon the organism, the tissue, and its environment. The importance of peroxisomes to humans is dramatically demonstrated by a family of lethal genetic diseases called Zellweger syndrome in which peroxisomes appear to be absent from or deficient in patient cells (Lazarow and Moser, 1989). Thus, peroxisomes are clearly essential for human survival. In recent years, basic features of peroxisome biogenesis have emerged. Proteins destined for the peroxisome are synthesized on free ribosomes, usually at their mature sizes, and posttranslationally imported into the organelle (Roa and Blobel, 1983; Fujiki et al., 1986). For import ofperoxisomal matrix enzymes, two distinct peroxisomal targeting signals (PTSs) ~ have been defined (for review see de Hoop and AB, 1992). The most common signal, PTS1, is a carboxyterminal tripeptide that is typically SKL or a conservative variant (Gould et al., 1987, 1990; Aitchison et al., 1991; Keller et al., 1991). The second, PTS2, is found on only a few peroxisomal enzymes such as mammalian and yeast 3-ketoacyl-CoA thiolases and yeast amine oxidase (Osumi et al., 1991; Swinkels et al., 1991; Glover et al., 1994; Faber et al., 1995). PTS2 is located near the amino terminus and 1. Abbreviations used in this paper: AMO, amine oxidase; AOX, alcohol oxidase; CAT, catalase; DHAS, dihydroxyacetone synthase; EM, electron microscopic; MAL, maltose binding protein; MOX, methanol oxidase; Mut +, methanol-utilizing; Mut-, methanol-utilization-defective; ORF, open reading frame; PMox, alcohol oxidase promoter; PTS, peroxisomal targeting signal. © The Rockefeller University Press, 0021-9525/95/02/307/13 $2.00 The Journal of Cell Biology, Volume 128, Number 3, February 1995 307-319 307 on Jne 9, 2007 w w w .jc.org D ow nladed fom is cleaved in some instances (rat thiolase) but not in others (yeast thiolase). Peroxisomal membrane proteins and some matrix enzymes do not appear to have either PTS; therefore, it is likely that one or more additional PTSs exist. Aside from targeting signals, import machinery components remain largely unidentified with the exception of the Pichia pastoris PAS8 gene product which may be a receptor for proteins bearing PTS1 (McCollum et al., 1993). Less is known regarding the cell's ability to control peroxisome volume and number. Like mitochondria, peroxisomes are thought to duplicate by fission from preexisting organelles and to actively migrate into daughter cells (Veenhuis et al., 1979; Attardi and Schatz, 1988). The number of peroxisomes per cell can increase dramatically in response to metabolic needs. For example, certain hypolipidemic drugs such as clofibrate induce peroxisomes to proliferate in rat liver cells (Lock et al., 1989). However, the most extreme example of a proliferative response is observed in methylotrophic yeasts such as Hansenula polymorpha where methanol induces a massive increase in peroxisome size and number (Veenhuis et al., 1979). In glucose-grown cells of this yeast, only one or a few small peroxisomes are present, whereas in methanol, peroxisomes can account for as much as 80% of total cell volume. Interestingly, when peroxisomal enzymes are expressed at high levels in glucose-grown cells, the organelles increase in size but not number (Gtdecke et al., 1989; Roggenkamp et al., 1989). Thus, it appears that peroxisome size may be a reflection of the amount of matrix protein, while proliferation is controlled by a separate mechanism. H. polymorpha is an attractive model system for genetic studies on peroxisome biogenesis (Veenhuis et al., 1992). In this organism, detailed physiological, biochemical, and ultrastructural information exists on the role of peroxisomes in the metabolism of a variety of unusual carbon and nitrogen sources (Veenhuis and Harder, 1987, 1991). In addition, methods for classicaland molecular-genetic manipulation of the organism are well developed (Cregg, 1987; Gleeson and Sudbery, 1988; Faber et at., 1992; Titorenko et al., 1993). In previous reports, we have described the isolation of H. polymorpha mutants that are defective in peroxisome biogenesis (per mutants) (Cregg et al., 1990; Waterham et al., 1992; Titorenko et al., 1993). In this report, we describe the characterization of one mutant, per8, and the molecular cloning and characterization of the PER8 gene and its product. We show that PER8 encodes a 34-kD peroxisomal integral membrane protein that appears to play a role in peroxisome proliferation. Materials and Methods Strains, Media, and Microbial Techniques H. polymorpha strains used in this study are listed in Table I. H. polymorpha cultures were grown at 37°C in a complex medium (YPD) composed of 1% yeast extract, 2 % peptone, and 2 % dextrose, or in one of the following minimal media: YNB medium (0.17% yeast nitrogen base without amino acids and ammonium sulfate, 0.5% ammonium sulfate) supplemented with either 0.4% dextrose or 0.5% methanol and 0.05% yeast extract; MAD medium (0.17% yeast nitrogen base without amino acids and ammonium sulfate, 0.25 % methylarnine, 0.25 % dextrose). Nutritional supplements for growth of auxotrophic strains were added to 50/~g/ml as required. Sporulation (mating) medium was composed of 2% malt extract. Table L H. polymorpha Strains Used in This Study Strain Genotype Source or reference CBS4732 wild type CBS4732 A16 leul Veale et al. (1992) C76 per8-1 This study CT100 per8-1 leul This study CT101 per8A: :SLEU2 This study CT102 per8A: :SLEU2 ade11 This study CT103 per8-1 leul PMox-PER8 This study CT104 perl leul This study Genetic manipulations ofH. polymorpha have been described (Gleeson and Sudbery, 1988; Cregg et at., 1990; Faber et al., 1992). Yeast transformations were performed by the Klebe procedure (KIebe et at., 1983). Cultivation of Escherichia coli strains and recombinant DNA techniques were performed as described (Sambrook et al., 1989).