Endoplasmic reticulum of yeast

The vesicles of endoplasmic reticulum of yeast cells contain PolyPs and also a specific system of their biosynthesis related to glycoproteins (Shabalin et al., 1979, 1985). 

Evidence that protein addition to GPI anchors occurs in the endoplasmic reticulum stems from kinetic studies on GPI anchor addition to newly synthesized proteins [97,98], the accumulation of unprocessed precursor proteins in the endoplasmic reticulum of yeast and mammalian mutants in GPI anchor biosynthesis [111,112], and the use of a microsomal assay system to analyze the C-terminal processing of GPI-linked proteins [113]. The biosynthesis of GPI anchors also is assumed to be localized to the endoplasmic reticulum. This assumption is well founded since GlcNAc-PI transferase and deacetylase activity co-fractionate with markers for the endoplasmic reticulum [114]. Therefore, at least the initial steps in GPI anchor biosynthesis occur in the endoplasmic reticulum. 

Suzuki, T., Park, H., Kitajima, K., and Lennarz, W. J. (1998). Peptides glycosylated in the endoplasmic reticulum of yeast are subsequently deglycosylated by a soluble peptide A-glycanase activity. J. Biol. Chem. 273, 21526-21530. 

Parodi, A. J., Reglucosylation of glycoproteins and quality control of glycoprotein folding in the endoplasmic reticulum of yeast cells, Biochim. Biophys. Acta, 1999, 1426, 287-295. 

Hagting A, Karlsson C, Clute P, Jackman M, Pines J (1998) MPF localization is controlled by nudear export. EMBO J 17 4127-4138 Hamman BD, Chen JC, Johnson EE, Johnson AE (1997) The aqueous pore through the translocon has a diameter of 40-60 A during cotranslational protein translocation at the ER membrane. Cell 89 535-544 Hampton RY, Rine J (1994) Regulated degradation of HMG-CoA reductase, an integral membrane protein of the endoplasmic reticulum, in yeast. J Cell Biol 125 299-312... 

Yeast and bacterial systems often give low levels of expression of silks, and this has led to the development of production systems in tobacco and potato. Scheller et al. (2001) have shown that spider silk proteins can be produced in transgenic plants. They inserted synthetic spider silk protein (spidroin) genes into transgenic plants under the control of the CaMV35S promoter. Using this system they were able to demonstrate the accumulation of recombinant silk proteins to a level of at least 2% of total soluble protein in the endoplasmic reticulum of tobacco leaves, and potato tubers. 

The endoplasmic reticulum of the yeast cell is a folded double-membrane structure which permeates the cytoplasm. The lumen between the membranes is 20 nm wide. From the reticulum, spherical vesicles develop, which can penetrate the plasmalemma and release their contents. Such vesicles appear to be involved in the synthesis of the cell wall, particularly that of developing buds. 

The cytochromes are iron-containing hemoproteins in which the iron atom oscillates between Fe + and Fe + during oxidation and reduction. Except for cytochrome oxidase (previously described), they are classified as dehydrogenases. In the respiratory chain, they are involved as carriers of electrons from flavoproteins on the one hand to cytochrome oxidase on the other (Figure 12-4). Several identifiable cytochromes occur in the respiratory chain, ie, cytochromes b, Cp c, a, and (cytochrome oxidase). Cytochromes are also found in other locations, eg, the endoplasmic reticulum (cytochromes P450 and h, and in plant cells, bacteria, and yeasts. 

The subcellular location of PG was studied in cells disrupted by osmotic lysis through formation and disruption of sphaeroplasts from self-induced anaerobically-grown cells. A discontinuous sucrose-density gradient produced four bands labelled I, II, III and IV. Band I included many vesicles and a peak of alkaline phosphatase activity (a vacuolar marker in yeasts), NADPH cytochrome c oxidoreductase activity, an endoplasmic reticulum marker, and... 

Boedallo, j. and D. H. Wole, A RING-H2 finger motif is essential For the function of Der3/Hrdl in endoplasmic reticulum associated protein degradation in the yeast Saccharomyces cerevisiae. FEBS Lett, 1999, 448(2-3), 244-8. 

Finger A, Knop M, Wolf, DH (1993) Analysis of two mutated vacuolar proteins reveals a degradation pathway in the endoplasmic reticulum or a related compartment of yeast. Eur J Biochem 218 565-74... 

Knop M, Hauser N, Wolf DH (1996a) N-Glycosylation affects endoplasmic reticulum degradation of a mutated derivative of carboxypeptidase yscY in yeast. Yeast 12 1229-1238... 

Mayer TU, Braun T, Jentsch, S (1998) Role of the proteasome in membrane extraction of a short-lived ER-transmembrane protein. EMBO ] 17 3251-3257 McCracken AA, Brodsky JL (1996) Assembly of ER-associated protein degradation in vitro dependence on cytosol, calnexin, and ATP. J Cell Biol 132 291-298 McDonald HB, Byers B (1997) A proteasome cap subunit required for spindle pole body duplication in yeast. J Cell Biol 137 539-553 McGee TP, Cheng HH, Kumagai H, Omura S, Simoni RD (1996) Degradation of 3-hydroxy-3-methylg utaryl-CoA reductase in endoplasmic reticulum membranes is accelerated as a result of increased susceptibility to proteolysis. J Biol Chem 271 25630-25638... 

Many secreted proteins, as well as smaller peptide hormones, are acted upon in the endoplasmic reticulum by tryptases and other serine proteases. They often cut between pairs of basic residues such as KK, KR, or RR.214-216 A substilisin-like protease cleaves adjacent to methionine.217 Other classes of proteases (e.g., zinc-dependent carboxypeptidases) also participate in this processing. Serine carboxypeptidases are involved in processing human prohormones.218 Among the serine carboxypeptidases of known structure is one from wheat219 and carboxypeptidase Y, a vacuolar enzyme from yeast.220 Like the pancreatic metallocarboxypeptidases discussed in Section 4, these enzymes remove one amino acid at a time, a property that has made carboxypeptidases valuable reagents for determination of amino acid sequences. Carboxypeptidases may also be used for modification of proteins by removal of one or a few amino acids from the ends. 

The biosynthesis in yeast of two enzymes that are D-mannoproteins has been studied. A membrane-associated isozyme of invertase (EC 3.2.1.26) has been shown to be a precursor of the external invertase.190 Its molecular weight, as determined by SDS-poly(acrylamide) gel electrophoresis, is 50,000, that is, smaller than that of the external invertase, and it correlates well with the presence of only the inner-core sugars of the final form. It is strictly bound to membranes, possibly those of the endoplasmic reticulum, and it can be completely split191 by endo-/3-N-acetylglucosaminidase H (EC 3.2.1.30). The addition of tunicamycin, which specifically inhibits formation of d-GIcNAc-PP-DoI, inhibits synthesis of external invertase, as well as further formation of the membrane-associated form, which completely disappears after addition of the antibiotic.190 In these aspects, the synthesis of this extracellular enzyme follows the pathway for secreted glycoproteins in animal systems. 

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