Characterization of New Mutants in the Early Part of the Yeast Secretory Pathway Isolated by a [ 3 H ] Mannose Suicide Selection

We have adapted a [3H]mannose suicide selection to identify mutations in additional genes which function in the early part of the yeast secretory pathway. Thus far this protocol has led to the identification of two new genes which are implicated in this process, as well as additional alleles of previously identified genes. The new mutants, bet1 and bet2, are temperature sensitive for growth and protein transport. Thin section analysis has revealed the accumulation of a network of endoplasmic reticulum (ER) at the restrictive temperature (37~ Precursors of exported proteins that accumulate in the cell at 37~ are terminally core glycosylated. These observations suggest that the transport of precursors is blocked subsequent to translocation into the ER but before entry into the Golgi apparatus. The betl and bet2 mutants define two new complementation groups which have the same properties as previously identified ER-accumulating mutants. This and previous findings (Novick, P., C. Field, and R. Schekman, 1980, Cell, 21:205-215) suggest that protein exit from the ER and entry into the Golgi apparatus is a complex process requiring at least 11 genes. A secreted, lysosomal, and membrane proteins initially use the same transport pathway. The early stages of protein export involve synthesis on cytoplasmic ribosomes, followed by targeting to and exit from the endoplasmic reticulum (ER). t Two components involved in the targeting of proteins to the ER have been identifiedsignal recognition particle and the signal recognition particle receptor, also called the docking protein (for review see Walter et al., 1984). Less is known about subsequent steps. After targeting, secretory proteins must be translocated through the ER membrane; then resident ER proteins must in some way be separated from those to be secreted. Studies demonstrating that different secretory proteins leave the ER at different rates (Fries et al., 1984; Lodish et al., 1983) suggest that these proteins also must be sorted from one another. Two hypotheses have been suggested to explain how this might occur. Blobel (1980) has proposed that protein exit from the ER can occur when specific protein sequences are recognized by their corresponding receptors. It has also been suggested (Kelly, 1985) that proteins move from the ER to the Golgi apparatus by bulk flow, except when specifically restrained or diverted. One such restraining protein, immunoglobulin heavy chain binding protein, has recently been identified (Bole et al., 1986). This binding protein has been shown to bind to immunoglobulin heavy chains and prevent 1. Abbreviations used in thispaper: CPY, carboxypeptidase Y; endo H, endoglycosidase H; ER, endoplasmic reticulum; ts, temperature sensitive. them from leaving the ER until they are complexed to light chains. To identify other proteins that mediate the interaction of exported proteins with the ER, we are following a genetic approach using the yeast, Saccharomyces cerevisiae. This simple eukaryote was shown to have a secretory pathway similar to that of mammalian cells (Novick et al., 1981). Using a technique that enriched for mutants that were denser than wild type at 37~ Novick et al. (1980) and Ferro-Novick et al. (1984) isolated a collection of yeast mutants that was temperature sensitive (ts) for growth and secretion. The mutants blocked export at four different stages of the yeast transport pathway: entry into the lumen of the ER, exit from the ER, transport out of the Golgi apparatus, and exocytosis. This enrichment required that ts mutants survive a 3-h incubation at T/~ This incubation was critical to the enrichment, since it was during this time that cells became dense. Therefore, any mutants that would die during such an incubation could not be selected. The density enrichment led to the isolation of a large number of mutants blocked at late stages of the secretory pathway. In contrast, fewer early blocked secretory mutants were identified. Mutations in only two genes, sec53 and sec59, blocked the completion of import of proteins into the ER (Ferro-Novick et al., 1984; Ferro-Novick, 1985). Mutations in nine genes (sec12, sec13, sec16, sec17, secl8, sec20, sec21, sec22, and sec23) affected transport subsequent to this but before entry into the Golgi apparatus (Novick et al., 1980); these were termed ER-accumulating mutants 9 The Rockefeller University Press, 0021-9525/87/10/1587/8 $2.00 The Journal of Cell Biology, Volume 105, October 1987 1587-1594 1587 on M ay 9, 2017 D ow nladed fom Published October 1, 1987