Intracellular Protein Transport 
Randy W. Schekman, Ph.D. — Investigator 
Dr. Schekman is also Professor of Biochemistry and Molecular Biology at the University of California, 
Berkeley, and Adjunct Professor of Biochemistry and Biophysics at the University of California, San 
Francisco. As a graduate student, he studied the enzymology ofDNA replication with Arthur Kornberg at 
Stanford University. His current interest in cellular membranes developed during a postdoctoral period 
with S. J. Singer at the University of California, San Diego. At Berkeley, he developed a genetic approach 
to the study of eukaryotic membrane trafftc. Among his awards is the American Society for Microbiology 
Eli Lilly Award in Microbiology and Immunology. Dr. Schekman was recently elected to the National 
Academy of Sciences. 
RESEARCH in our laboratory is devoted to mo- 
lecular description of the processes of poly- 
peptide translocation from the cytosol into the 
endoplasmic reticulum (ER) and of vesicular 
transport among organelles of the secretory 
pathway. 
Genetic and Biochemical Dissection 
of the Secretory Process 
A genetic approach to the study of eukaryotic 
protein transport involved the isolation of condi- 
tional mutants. We isolated a series of secretory 
{sec) mutants in the yeast Saccharomyces cerevi- 
siae that are temperature-sensitive for cell sur- 
face growth, division, and secretion. Most of the 
mutants accumulate secretory proteins in an in- 
tracellular pool that can be released when cells 
are returned to a permissive temperature. More 
than 30 gene products have been implicated in 
the process of delivering membrane and secre- 
tory proteins to the cell surface. 
A combined genetic and cytologic evaluation 
of the sec mutants has allowed a description of 
the secretory pathway. Protein transport in yeast 
appears to be mediated by the same organelles 
and proteins that operate in mammalian cells. Mo- 
lecular cloning analysis of SEC genes has re- 
vealed striking structural and functional homol- 
ogy with corresponding mammalian genes. 
We have developed biochemical assays that 
measure the early events of polypeptide translo- 
cation into the ER and of vesicle-mediated pro- 
tein transport from the ER to the Golgi apparatus. 
The cell-free reactions represent physiologically 
meaningful processes. Extracts prepared from 
mutant cells reproduce the temperature-sensitive 
defects observed in vivo. In favorable circum- 
stances the mutant defects are repaired by addi- 
tion of a protein fraction obtained from wild-type 
yeast, and such restoration of transport activity 
may be used to purify functional forms of SEC 
gene products. 
Protein Translocation Across Membranes 
Protein translocation into the lumen of the en- 
doplasmic reticulum represents the initial step in 
assembly of the eukaryotic cell surface. This pro- 
cess has been reconstituted with detergent-solubi- 
lized membrane proteins and purified cytosolic 
proteins, yet the mechanism of polypeptide pen- 
etration is unclear. We have isolated mutants that 
are defective in translocation, using a genetic se- 
lection that requires secretory polypeptides to be 
retained in the cytosol. The work on these mu- 
tants is supported by a grant from the National 
Institutes of Health and will not be described 
here. 
An independent line of investigation concerns 
the mechanism of protein translocation from the 
cytosol into the vacuole. The vacuole contains an 
array of hydrolytic enzymes and is believed to 
play an important role in the degradation of cyto- 
solic proteins and intracellular membranes. The 
mechanism for importing substrates into the vacu- 
ole has not been evaluated. 
Last year we reported a novel pathway for the 
localization and degradation of fructose 1,6-bis- 
phosphatase (FBPase), a key regulatory enzyme 
of gluconeogenesis. FBPase is localized to the cy- 
tosol when cells are grown on a poor carbon 
source, such as ethanol. When cells are trans- 
ferred to glucose, FBPase is degraded in a process 
called catabolite inactivation, which depends on 
active vacuolar proteases. In a protease-deficient 
strain, FBPase enters the vacuole and remains in- 
tact. Import into the vacuole was shown to de- 
pend on protein synthesis during the period of 
transfer to glucose medium. In addition, vacuolar 
import requires the transfer of a protein, possibly 
an import receptor, via the secretory pathway. 
The mechanism of this new import pathway is 
being pursued by the isolation of mutants defec- 
tive in the degradation of FBPase. Thus far a large 
number of genes have been identified that are 
required for import of FBPase into the vacuole. 
Preliminary evidence suggests that the import 
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