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Abstract. The YF//gene encodes a raslike, GTPbinding protein that is essential for growth of yeast cells. We show here that mutations in the yptl gene

Abstract. Using either permeabilized cells or microsomes we have reconstituted the early events of the yeast secretory pathway in vitro. In the first stage of the reaction ,'o50-70% of the prepro-ct-factor, synthesized in a yeast translation lysate, is translocated into the endoplasmic reticulum (ER) of permeabilized yeast cells or directly into yeast microsomes. In the second stage of the reaction 48-66 % of the ER form of a-factor (26,000 D) is then converted to the high molecular weight Golgi form in the presence of ATP, soluble factors and an acceptor membrane fraction; GTPyS inhibits this transport reaction. Donor, acceptor, and soluble fractions can be separated in this assay. This has enabled us to determine the defective fraction in sec23, a secretory mutant that blocks ER to Golgi transport in vivo. When fractions were prepared from mutant cells grown at the permissive or restrictive temperature and then assayed in vitro, the acceptor Golgi fraction was found to be defective.

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Abstract.We have used an in vitro assay that reconstitutes transport from the ER to the Golgi complex in yeast to identify a functional vesicular intermediate in transit to the Golgi apparatus. Permeabilized yeast cells, which serve as the donor in this assay, release a homogeneous population of vesicles that are biochemically distinct from the donor ER fraction. The isolated vesicles, containing a post-ER/pre-Golgi form of the marker protein pro-a-factor, were able to bind to and fuse with exogenously added Golgi membranes. The ability to isolate fusion competent vesicles provides direct evidence that ER to Golgi membrane transport is mediated by a discrete population of vesicular carriers.

We have cloned an 82-base-pair region spanning the site of normal 3' end formation of Saccharomyces cerevisiae CYCI mRNA into an integrative vector carrying the 5' end of the actin gene (including its intron) fused in frame to HIS4ABC sequences. This vector can confer the ability to grow on histidinol if HIS4C (encoding histidinol dehydrogenase) is sufficiently expressed. With the CYCI fragment cloned in its wild-type (forward) orientation within the actin intron, transformants cannot grow on histidinol, whereas cells transformed with the vector carrying the reverse orientation ofthis fragment are able to grow well. RNA transfers demonstrate that transformants containing the forward orientation accumulate <40% of the control level of full-length mRNA and reveal the presence ofa short, stable (=300 nucleotides) poly(A) RNA that represents 60-70% ofthe transcripts originating from the same promoter. The reverse orientation of the insert allows nearnormal levels offull-length mRNA. Mapping ofthe 3' end ofthe truncated RNA indicates that poly(A) addition is variable in length but occurs at the same location as in the normal CYCI transcript. Dominant and recessive suppressor mutations permit growth on histidinol despite the inserted fragment. Genetic analyses indicate that most of the dominant mutants are cis-acting and that the recessive mutants define a minimum of three complementation groups, indicating that defects in several different genes can restore higher levels of HIS4C expression.Sherman (3) when they found that the cycl-512 mutation [a 38-base-pair (bp) deletion spanning the wild-type CYCI mRNA 3' end point] resulted in a 90%o reduction in the overall accumulation oftranscripts encoding the CYCI product; many of the mRNAs were longer, and all were polyadenylylated. Several other yeast genes share limited homology to a subset of the sequences deleted by the cycl-512 mutation. Local sequence changes in these regions can restore partial function (4-6); however, no definitive sequence has yet been proved to specify 3' end formation. Analysis of cycl-512 revertants demonstrated that mutations in trans-acting factors can also act to restore the efficiency of 3' end formation and accumulation of stable mRNAs (4, 7).In this report we describe a combined genetic and biochemical approach to understanding 3' end formation and polyadenylylation of yeast mRNA. We have used an in-frame fusion of the actin and HIS4 genes (8) (8); genetic markers include ura3-52 for selection of URA3 transformants, his4-401 [a 2-kilobase (kb) deletion encompassing the promoter and most of the coding sequences], and the dominant HOLI mutation to permit uptake of the histidine precursor, histidinol. In addition, either ade2 or ade5 markers were introduced into the strains to permit selection of diploids used in complementation tests. Yeast were transformed after spheroplasting (9), and other standard genetic techniques and growth media were as described by Sherman et al. (10). In SC + Hol medium, 0.064% histidinol was included in s...

The sec53 mutant is a conditional lethal yeast secretory mutant. At 37°C, precursors to exported proteins become firmly attached to the endoplasmic reticulum membrane and are not released into the lumen in a soluble form. The accumulated precursors are insoluble in the detergent Triton X-100; however, urea, a known protein denaturant, solubilizes them. Using antibody directed against the Sec53 protein, we found that a substantial portion of the Sec53 protein is associated with the cytoplasmic surface of the endoplasmic reticulum membrane. Membrane-bound SecS3 protein is largely insoluble in Triton X-100, but the protein is effectively released from the membrane by urea. We propose that the SecS3 protein may be a member of a complex of proteins required for an early step in protein processing and transport.A biochemical dissection of heterologous in vitro translocation systems in higher eukaryotes has led to the identification of two components that play a role in targeting proteins to the endoplasmic reticulum (ER): signal recognition particle and docking protein, also called the signal recognition particle receptor (1-3). Based on in vitro findings the signal recognition particle has been proposed to recognize the signal sequence of exported proteins and target them to the signal recognition particle receptor, which resides on the ER membrane. Translocation across the bilayer is believed to occur through an aqueous tunnel (4).Genetic studies in yeast have identified two genes, SEC53 and SEC59, whose products have been implicated in protein translocation across the ER membrane (5, 6). These mutants rapidly and dramatically block the transit of all major cellsurface proteins at the restrictive temperature of 37°C, but not at the permissive temperature of 24°C. DNA sequence analysis has shown that the SEC53 gene product is a hydrophilic protein; hydropathic analysis has not revealedany hydrophobic regions long enough to span the lipid bilayer (7). In this report we show that two pools of Sec53 protein (Sec53p) exist in wild-type cells, a soluble fraction and a membrane-bound fraction. Membrane-bound Sec53p is attached to the cytoplasmic surface of the ER membrane. Our data suggest that this localization is mediated by a protein-protein interaction. The precursors to exported proteins that accumulate in the secS3 mutant at 37°C also appear to adhere to the ER membrane as a result of a protein-protein interaction.MATERIALS AND METHODS Buffers. Phosphate-buffered saline (PBS) is 0.2 M sodium chloride/12.5 mM potassium phosphate, pH 7.6. Dilution buffer is PBS with 2% Triton X-100 plus aprotinin (100 units/ml). Spheroplast medium is 1.4 M sorbitol/50 mM potassium phosphate, pH 7.5/10 mM sodium azide/56 mM pB-mercaptoethanol containing zymolyase (1 mg/ml). Sorbitol cushion is 1.7 M sorbitol/20 mM sodium phosphate, pH 7.5. Lysis buffer is 0.3 M mannitol/10 mM Mops, pH 7.0, containing N,N'-diphenyl-p-phenylenediamine (0.1 pug/ml), chymostatin (1 ,ug/ml), and aprotinin (100 units/ml). and washed once with 10 mM so...

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