Two members of the hsp70 family, termed hsc70 and BiP, have been implicated in promoting protein folding and assembly processes in the cytoplasm and the lumen of the endoplasmic reticulum, respectively. Short hydrophilic (8 to 25 residues) synthetic peptides have now been tested as possible mimics of polypeptide chain substrates to help define an enzymatic basis for these activities. Both BiP and hsc70 have specific peptide binding sites. Peptide binding elicits hydrolysis of adenosine triphosphate, with the subsequent release of bound peptide.

We carried out a test sample study to try to identify errors leading to irreproducibility, including incompleteness of peptide sampling, in LC-MS-based proteomics. We distributed a test sample consisting of an equimolar mix of 20 highly purified recombinant human proteins, to 27 laboratories for identification. Each protein contained one or more unique tryptic peptides of 1250 Da to also test for ion selection and sampling in the mass spectrometer. Of the 27 labs, initially only 7 labs reported all 20 proteins correctly, and only 1 lab reported all the tryptic peptides of 1250 Da. Nevertheless, a subsequent centralized analysis of the raw data revealed that all 20 proteins and most of the 1250 Da peptides had in fact been detected by all 27 labs. The centralized analysis allowed us to determine sources of problems encountered in the study, which include missed identifications (false negatives), environmental contamination, database matching, and curation of protein identifications. Improved search engines and databases are likely to increase the fidelity of mass spectrometry-based proteomics.

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Strains of the species Komagataella phaffii are the most frequently used “Pichia pastoris” strains employed for recombinant protein production as well as studies on peroxisome biogenesis, autophagy and secretory pathway analyses. Genome sequencing of several different P. pastoris strains has provided the foundation for understanding these cellular functions in recent genomics, transcriptomics and proteomics experiments. This experimentation has identified mistakes, gaps and incorrectly annotated open reading frames in the previously published draft genome sequences. Here, a refined reference genome is presented, generated with genome and transcriptome sequencing data from multiple P. pastoris strains. Twelve major sequence gaps from 20 to 6000 base pairs were closed and 5111 out of 5256 putative open reading frames were manually curated and confirmed by RNA-seq and published LC-MS/MS data, including the addition of new open reading frames (ORFs) and a reduction in the number of spliced genes from 797 to 571. One chromosomal fragment of 76 kbp between two previous gaps on chromosome 1 and another 134 kbp fragment at the end of chromosome 4, as well as several shorter fragments needed re-orientation. In total more than 500 positions in the genome have been corrected. This reference genome is presented with new chromosomal numbering, positioning ribosomal repeats at the distal ends of the four chromosomes, and includes predicted chromosomal centromeres as well as the sequence of two linear cytoplasmic plasmids of 13.1 and 9.5 kbp found in some strains of P. pastoris.

We have cloned a gene encoding an alpha 1,2 galactosyltransferase activity from Schizosaccharomyces pombe. The open reading frame of the gene (gma12 for galactomannan, alpha 1,2), combined with the previous protein purification (Chappell and Warren, 1989), predicts an O-linked glycoprotein with type II transmembrane topology. By homologous gene disruption, we have demonstrated that the gma12 gene product (gma12p) is nonessential. The deletion strain (gma12-D10::ura4) has a significantly reduced level of galactosyltransferase activity relative to the parental strain, but both in situ lectin binding and in vitro biochemical assays demonstrate the presence of further galactosyltransferase activity in addition to gma12p. Although gma12p is not the only galactosyltransferase in S. pombe, it produces a unique carbohydrate structure on the surface of the yeast cells. We have generated a polyclonal antiserum against this carbohydrate epitope and shown that gma12p is capable of synthesizing the epitope both in vitro and in vivo. Electron microscopic localization of the gma12+ specific epitope in gma12+ cells revealed that gma12p synthesizes the carbohydrate structure in the Golgi apparatus, and subsequent intracellular transport distributes the epitope to later stages of the secretory pathway. The immunolocalization studies confirm the presence of one or more galactosyltransferase activities in the Golgi apparatus in fission yeast.

Abstract. A membrane-associated galactosyltransferase has been purified to homogeneity from the fission yeast, Schizosaccharomyces pombe. The enzyme has a molecular weight of 61,000 and is capable of transferring galactose from UDP-galactose (UDP-Gal) to a variety of mannose-based acceptors to form an or-l,2 galactosyl mannoside linkage. Immunofluorescence localization of the protein is consistent with the presence of the enzyme in the Golgi apparatus of S. pombe. This, together with the presence of terminal, or-linked galactose on the N-linked oligosaccharides of S.pombe secretory proteins, suggests that the galactosyltransferase is an enzyme involved in the processing of glycoproteins transported through the Golgi apparatus in fission yeast. Is recent years, two complementary approaches have been taken to identify components involved in protein transport from the endoplasmic reticulum, through the Golgi apparatus, to the cell surface. The genetic approach, exemplified by Schekman and his co-workers, has identified several classes of gene products involved at different stages of protein secretion in Saccharomyces cerevisiae, from initial translocation and glycosylation to final delivery to the cell surface (for review, see Schekman, 1985). The use of cell-free systems, initiated by Rothman, has increased our understanding of the biochemical mechanisms involved in vesicular traffic, primarily through the Golgi apparatus of mammalian cells (for review see Pfeffer and Rothman, 1987). The demonstration that yeast contains all the cytosolic components required for mammalian intra-Golgi transport (Dunphy et al., 1986) and the recent finding that the sec18 gene product of S. cerevisiae is functionally equivalent to one of the purified components of the Golgi transport system (N-ethylmaleimide-sensitive factor) (Wilson et al., 1989) demonstrate the efficacy of a combined genetic and biochemical approach to the questions of vesicular trafficking.The primary method for tracing the progress of a protein through the transport pathway is to follow the processing of N-linked oligosaccharides. In the endoplasmic reticulum of mammalian cells, N-linked Glc3MangGlcNAc2 oligosaccharides are first trimmed to a MansGlcNAc2 core, and then, as the glycoprotein is transported through the Golgi apparatus, processed by a series of mannosidases and sugar transferases to a final complex structure containing mannose, additional GIcNAc, galactose (Gal) t and sialic acid (for review, see Kornfeld and Kornfeld, 1985). Elucidation of the pathway of N-linked oligosaccharide processing and immunolocalization of some of the enzymes involved have demonstrated 1. Abbreviations used in this paper: Gal, galactose; Glc, glucose; GIcNAc, N-acetyl-glucosarnine; Man, mannose. that the Golgi apparatus in mammalian cells consists of distinct biochemical compartments through which secretory, lysosomal, and plasma membrane proteins pass in a vectorial fashion (for review, see Dunphy and Rothman, 1985). The relatively simple processing of N-linked oligosac...

During oocyte development in Caenorhabditis elegans, approximately half of all developing germ cells undergo apoptosis. While this process is evolutionarily conserved from worms to humans, the regulators of germ cell death are still largely unknown. In a genetic screen for novel genes involved in germline apoptosis in Caenorhabditis elegans, we identified and cloned gla-3. Loss of gla-3 function results in increased germline apoptosis and reduced brood size due to defective pachytene exit from meiosis I. gla-3 encodes a TIS11-like zinc-finger-containing protein that is expressed in the germline, from the L4 larval stage to adulthood. Germ stem cells are the precursors to all subsequent generations in a species, and, therefore, germ cell formation is tightly monitored to ensure high-fidelity transfer of the genetic material. Germ cell genome integrity is monitored at several checkpoints, allowing for DNA repair, cell cycle arrest, and apoptosis when required. In mammals, inactivation of genes with checkpoint function frequently results in aberrant cell death and infertility (Lim and Hasty 1996;Bender et al. 2002). Germ cell development is also characterized by either massive waves or low but constant levels of apoptosis that are not caused by genetic defects. For example, >99.9% of oocytes undergo apoptosis in response to hormonal changes that occur at several stages during the female life cycle in mammals (Tilly 2001;Kim and Tilly 2004). Apoptosis is also the fate of ∼50% of germ cells undergoing oogenesis in the gonad of Caenorhabditis elegans hermaphrodites. (Gumienny et al. 1999). Characterization of the pathways that regulate germ cell death will contribute to our understanding of the cell suicide decision and might allow for more efficient therapeutic manipulation of the apoptotic program.C. elegans is a good model to study the signaling cascades involved in the decision between germ cell survival and germ cell death (Hengartner 1997;Gumienny et al. 1999). The adult hermaphrodite gonads consist of two U-shaped tubes that are connected at a common uterus. At the distal end of each gonad, mitotic germ stem cells proliferate in response to the Notch ligand LAG-2. Cells beyond the influence of LAG-2 enter meiosis and progress through the pachytene stage of meiosis I; this transition requires activation of the RAS/MAPK (MAP kinase) signaling cascade (Hubbard and Greenstein 2000;Seydoux and Schedl 2001). Following transition through pachytene, germ cells can either enter diakinesis of meiosis I and differentiate into oocytes or undergo apoptosis. We previously suggested that these cell deaths are the result of a physiological, homeostatic control mechanism that limits the number of germ cells permitted to differentiate into oocytes (Gumienny et al. 1999).