Abstract. The Saccharomyces cerevisiae APE1 gene product, aminopeptidase I (API), is a soluble hydrolase that has been shown to be localized to the vacuole. API lacks a standard signal sequence and contains an unusual amino-terminal propeptide. We have examined the biosynthesis of API in order to elucidate the mechanism of its delivery to the vacuole. API is synthesized as an inactive precursor that is matured in a PEP4-dependent manner. The half-time for processing is approximately 45 min. The API precursor remains in the cytoplasm after synthesis and does not enter the secretory pathway, The precursor does not receive glycosyl modifications, and removal of its propeptide occurs in a sec-independent manner. Neither the precursor nor mature form of API are secreted into the extracellular fraction in Ws mutants or upon overproduction, two additional characteristics of soluble vacuolar proteins that transit through the secretory pathway. Overproduction of API results in both an increase in the half-time of processing and the stable accumulation of precursor protein. These results suggest that API enters the vacuole by a posttranslational process not used by most previously studied resident vacuolar proteins and will be a useful model protein to analyze this alternative mechanism of vacuolar localization.

We have carried out a screen of 622 deletion strains generated during the EUROFAN B0 project to identify non-essential genes related to the mannosylphosphate content of the cell wall. By examining the affinity of the deletants for the cationic dye alcian blue and the ion exchanger QAE-Sephadex, we have selected 50 strains. On the basis on their reactivity (blue colour intensity) in the alcian blue assay, mutants with a lower phosphate content than wild-type cells were then arranged in groups defined by previously characterized mutants, as follows: group I (mnn6 ), group II (between mnn6 and mnn9 ) and group III (mnn9 ). Similarly, strains that behaved like mnn1 (i.e. a blue colour deeper than wild-type) were included in group VI. To confirm the association between the phenotype and a specific mutation, strains were complemented with clones or subjected to tetrad analysis. Selected strains were further tested for extracellular invertase and exoglucanase. Within groups I, II and III, we found some genes known to be involved in oligosaccharide biosynthesis (ALG9, ALG12, HOC1 ), secretion (BRE5, COD4/COG5, VPS53 ), transcription (YOL072w/THP1, ELP2, STB1, SNF11 ), cell polarity (SEP7, RDG1 ), mitochondrial function (YFH1 ), cell metabolism, as well as orphan genes. Within group VI, we found genes involved in environmentally regulated transduction pathways (PAL2 and RIM20 ) as well as others with miscellaneous or unknown functions. We conclude that mannosylphosphorylation is severely impaired in some deletants deficient in specific glycosylation/secretion processes, but many other different pathways may also modulate the amount of mannosylphosphate in the cell wall.

N-oligosaccharides of Saccharomyces cerevisiae glycoproteins are classified as core and mannan types. The former contain 13-14 mannoses whereas mannan-type structures consist of an inner core extended with an outer chain of up to 200 -300 mannoses, a process known as hyperglycosylation. The selection of substrates for hyperglycosylation poses a theoretical and practical question. To identify hyperglycosylation determinants, we have analyzed the influence of the second amino acid (Xaa) of the sequon in this process using the major exoglucanase as a model. Our results indicate that negatively charged amino acids inhibit hyperglycosylation, whereas positively charged counterparts promote it. On the basis of the tridimensional structure of Exg1, we propose that Xaa influences the orientation of the inner core making it accessible to mannan polymerase I in the appropriate position for the addition of ␣-1,6-mannoses. The presence of Glu in the Xaa of the second sequon of the native exoglucanase suggests that negative selection may drive evolution of these sites. However, a comparison of invertases secreted by S. cerevisiae and Pichia anomala suggests that hyperglycosylation signals are also subjected to positive selection.Protein glycosylation in eukaryotic cells is thought to play an essential role in many processes such as protein folding and transport, maintenance of protein and cell structure, and cell recognition and adhesion, as well as other functions. From the several types of protein glycosylation, N-glycosylation has received a great deal of attention not only because of its high frequency but also because several biochemical steps involved in this biosynthetic process are shared by yeast and humans, an indication that they have been conserved throughout evolution. These conserved steps occur in the membrane (i) or the lumen (ii and iii) of the ER 1 and belong to three groups: (i) assembly of the precursor oligosaccharide, GlnNAc 2 -Man 9 Glc 3 on a lipid carrier (dolichol-PP), (ii) transfer of the oligosaccharide to the nascent or recently synthesized protein acceptor, and (iii) trimming of the three glucoses and one mannose (for recent reviews, see Refs. 1 and 2).However, once the glycoprotein leaves the ER, biochemical modification by trimming and/or addition of new sugars varies enormously between species and even between individual proteins of the same cell. This suggests that individual proteins carry the precise information for the final carbohydrate composition. In Saccharomyces cerevisiae, some of the protein-attached oligosaccharides leaving the ER (GlcNAc 2 -Man 8 ) are poorly elongated with up to 13-14 mannoses (coretype), whereas many others are further elongated by the addition of an outer chain of up to 200 mannose residues in the Golgi apparatus (mannan-type), a process commonly known as hyperglycosylation. The outer chain consists of a backbone of ␣-1,6-mannoses with ␣-1,2 branches that are decorated with terminal ␣-1,3-mannose residues (1, 3). The biosynthesis of this complex is carried out...

The structural gene, APEl, (LAP4), for the vacuolar aminopeptidase I of Saccharomyces cerevisiae was cloned with the aid of a staining technique which permitted monitoring of aminopeptidase activity in yeast colonies. Genetic linkage data demonstrate that integrated copies of the cloned gene map to the APE1 locus. The nucleotide sequence of the cloned gene was determined. The open reading frame of APE1 consists of 514 codons and, therefore, encodes a larger protein (MW 57 003) than the reported mature aminopeptidase ysc1 (MW 44 SOO), suggesting that proteolytic processing must occur. A 1.75-kb mRNA, which is made in substantial amounts only when yeast cells have exhausted the glucose supply, was identified.

Plasmids capable of complementing lupl, lap2 and lap3 mutations [R. J. Trumbly and G. Bradley (1983) J. Bucteriol. 156, [36][37][38][39][40][41][42][43][44][45][46][47][48] were isolated from a yeast YEpl3 library by screening for activity against the chromogenic aminopeptidase substrate L-leucine P-naphthylamide in intact yeast colonies. The genomic inserts were shown to contain the structural genes for aminopeptidases yscII, yscIII and ysclV. Plasinids containing the gene encoding aminopeptidase yscII of Sacchuromyce.y cerevisiae, APE2 (LAPI) were analyzed in detail. APE2 was determined by DNA blot analysis to be a singlecopy gene located on chromosome XI. The cloned fragment was used to identify a 2.7-kb mRNA. The proteolytic system of the yeast Succhuromyces cerrvihiue has attracted considerable attention and is still actively studied (for reviews, see [I, 21). The yeast proteinases located in the vacuole, the lysosome-like organelle [3], are by far the best characterized. The genes encoding proteinase yscA, proteinase yscB, aminopeptidase yscI, dipeptidyl aminopeptidase yscV, carboxypeptidase yscY and carboxypeptidase yscS have been cloned and sequenced [4-221 and their biosynthesis and sorting have been examined (reviewed in [13]).Matile et al. [14] detected four aminopeptidases with activity on leucine /3-naphthylamide after separating crude extracts from yeast by starch gel electrophoresis. Four aminopeptidases capable of hydrolyzing tripeptides were deCorrespondeelwe to P. SuCrez-Rendueles,