Protein Targeting

Proteins coded by the nuclear genome are synthesized in the cytoplasm and must be delivered to different membrane-bound organelles within the cell or secreted outside. How does the cell accomplish this? S. cerevisiae played a leading role in identifying the major players in this cellular choreography and early conceptual breakthroughs that came from mutant hunts and genetic analysis.

To identify the genes which, when mutated, would cause a defect in protein secretion, Peter Novick and Randy Schekman used two marker proteins, phosphatase and invertase, whose secretion is easily detected by simple colorimetric assays (Novick and Schekman, 1979; Novick et al., 1980). Yeast cells were mutagenized and plated. Colonies were selected that were defective in the secretion of both enzymes at the restrictive temperature 37°C but not at permissive temperatures of 22 to 24°C. The mutants obtained were organized into 23-complementation groups, suggesting participation of at least 23 genes involved in the secretory pathway. The ts mutants are tools to examine the function of essential genes; in addition, they facilitate capturing and analyzing intermediate steps in complex biological processes by imposing the defect at the restrictive temperature. For example, at the restrictive temperature, SEC18 mutant accumulates 50-nm vesicles containing proteins with pattern of glycosylation specific to the endoplasmic reticulum. This suggests that the SEC18 gene product functions in the fusion of the ER-derived vesicles with the Golgi-membrane. Many of the proteins involved in vesicular transport identified by biochemical studies of mammalian cell-free systems have been confirmed by genetic approaches using yeast.

The mutants were analyzed to define the order of events in the secretory pathway by the method of double mutant analysis first employed by L. H. Hartwell in 1974 to describe the sequence of events in the yeast cell cycle. Briefly, electron microscopic analysis of the mutants belonging to each of the 23-complementation groups indicated that the mutants specifically accumulated three different membrane-enclosed structures when shifted to the non-permissive temperature. They were either endoplasmic reticulum

Figure 6.9 Use of yeast mutants in identification of steps in protein secretion. (From Lodish, H. et al., Molecular Cell Biology, © 1995, Scientific American Books, Inc. With permission from W.H. Freeman Company.)

structures or cup-shaped structures called "Berkeley bodies" or 80 to 100 nm vesicles. Very rarely a single mutant showed over-representation of more than one structure, suggesting that each mutant is blocked at a discrete step in the process. In double mutant analysis, two mutants that accumulate different structures are combined and its organellar structure is analyzed. It is expected that a double mutant would accumulate a structure that corresponds to the earliest block. Using this method, the secretory mutants (sec-mutants) were placed along a linear pathway reflecting the major steps in the secretory process (Figure 6.9).

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