Intracellular Transport of Proteins 
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 Fran- 
cisco. As a graduate student, he studied the enzymology of DNA 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 traffic. 
RESEARCH in our laboratory is devoted to a 
molecular description of two processes: 
polypeptide translocation from the cytosol into 
the endoplasmic reticulum (ER) and vesicular 
transport among organelles of the secretory 
pathway. 
Genetic Dissection of the Secretory Process 
A genetic approach to the study of protein 
transport in eukaryotes involved the isolation of 
conditional mutants. We isolated a series of se- 
cretory {sec) mutants in the yeast Saccharo- 
myces cerevisiae that are temperature-sensitive 
for cell surface growth, division, and secretion. 
Most of the mutants accumulate secretory pro- 
teins in an intracellular pool that can be released 
when cells are returned to a permissive tempera- 
ture. Over 30 gene products have been impli- 
cated in the process of delivering membrane and 
secretory proteins to the cell surface. 
A combination of genetic and cytologic evalua- 
tion of the sec mutants has allowed a description 
of the secretory pathway. Protein transport in 
yeast appears to be mediated by the same organ- 
elles and proteins that operate in mammalian 
cells. Molecular cloning analysis of 5.£'C genes has 
revealed striking structural and functional homol- 
ogy with corresponding mammalian genes. 
Protein Translocation Into 
the Endoplasmic Reticulum 
Protein translocation into the lumen of the ER 
represents the initial step in assembly of the eu- 
karyotic cell surface. This process has been re- 
constituted with detergent-solubilized mem- 
brane proteins and purified cytosolic proteins, 
yet the mechanism of polypeptide penetration is 
unclear. We have isolated mutants that are defec- 
tive in translocation, using a genetic selection 
that requires secretory polypeptides to be re- 
tained in the cytosol. 
Four SEC genes have been identified whose 
gene products are required for the translocation 
of a wide variety of secretory and membrane pro- 
teins. Three of the Sec proteins (Sec61p, -62p, 
and -63p) are found in a complex that may be 
isolated from detergent-solubilized membranes. 
The complex includes two other polypeptides of 
unknown structure. This complex may catalyze 
the translocation event or create a pore in the ER 
membrane through which secretory polypep- 
tides are transmitted. 
At least one of the proteins in the translocation 
complex forms a direct contact with the nascent 
secretory polypeptide. A protein conjugate con- 
sisting of the yeast secretory protein, a-factor 
precursor, linked chemically to avidin, engages 
but jams the translocation apparatus. Treatment 
of the jammed complex with a cleavable cross- 
linking reagent generates a covalent connection 
between a-factor precursor and Sec61p. The re- 
quirements for the production of this interme- 
diate suggest that the contact between Sec6lp and 
the secretory polypeptide follows an earlier 
event in which a-factor first binds to a receptor 
on the cytosolic surface of the ER membrane. 
A fourth gene, SEC65, required for transloca- 
tion, resembles the 19-kDa subunit of the mam- 
malian signal recognition particle (SRP). sec65 
mutants are defective in the translocation of mole- 
cules that are inserted cotranslationally, such as 
yeast invertase, or post-translationally, such as a- 
factor precursor. This implies that at least part of 
a yeast SRP panicipates in the translocation of 
molecules uncoupled from ongoing protein 
synthesis. 
Two forms of the 70-kDa heat-shock protein 
family (heat-shock cognate, orHscVO) also partic- 
ipate in the translocation event. Depletion of cy- 
tosolic Hsc70 by genetic manipulation results in 
the accumulation of certain secretory and mito- 
chondrial precursor proteins. A direct participa- 
tion of these molecules in the translocation event 
was established by reconstitution of Hsc70- 
dependent in vitro a-factor precursor assembly 
into yeast ER vesicles. Cytosolic Hsc70 may asso- 
ciate with translocation precursor polypeptides 
and prevent them from folding into unfavorable 
structures or complexes. 
A luminal Hsc70, homologous to mammalian 
BiP, was shown by Joe Vogel and Mark Rose at 
Princeton also to be involved in translocation. 
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