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MECHANISM OF INTRACELLULAR PROTEIN TRANSPORT 
Randy W. Schekman, Ph.D., Investigator 
Research in Dr. Schekman's laboratory is focused 
on the molecular mechanism of protein traffic in the 
secretory pathway. A combined genetic and bio- 
chemical approach was developed to study this 
pathway in Saccharomyces cerevisiae. The isola- 
tion of a large number of mutations that define the 
secretory pathway and the characterization of the 
affected gene products have demonstrated that the 
pathway and the molecular machinery of protein 
transport are highly conserved among eukaryotes. A 
cell-free reaction that reproduces roughly the first 
half of the secretory pathway is now being used to 
isolate functional forms of the cytosolic and mem- 
brane-bound proteins of the secretory machinery. 
Vesicle-mediated Protein Transport 
from the Endoplasmic Reticulum 
More than 20 SEC genes are required for the for- 
mation, targeting, and fusion of small vesicles that 
carry proteins from the endoplasmic reticulum (ER) 
to the Golgi apparatus. Cytologic evaluation of mu- 
tant cells has allowed the assignment of roles in vesi- 
cle budding and targeting to seven of these genes. A 
more thorough analysis of the role of the complete 
set of gene products involved in this limb of the 
secretory pathway has become possible through the 
development of a cell-free system that reproduces 
the transport-dependent glycosylation of in vitro 
synthesized a-factor precursor. This reaction is 
quantified by precipitation of the radioactive prod- 
uct, using antibodies directed against carbohydrate 
epitopes that are covalently attached to the glyco- 
protein substrate in the Golgi apparatus. Transport 
is dependent on ATP, GTP hydrolysis, cytosol, and 
intact membranes. The radioactive marker is trans- 
ferred between physically separable organelles (ER 
and Golgi) via an intermediate vesicle that has dis- 
tinctive properties. Transport is temperature sensi- 
tive in extracts of certain thermosensitive sec mu- 
tant strains and is inhibited by antibodies directed 
against recombinant or native forms of Sec proteins. 
A simplified assay has been developed that mea- 
sures only the vesicle budding reaction. The a- 
factor precursor, trapped within the ER, sediments 
rapidly, whereas when vesicle transport begins the 
precursor appears within slowly sedimenting vesi- 
cles. Budding can thus be followed simply by differ- 
ential centrifugation of lysates. This reaction has al- 
lowed the isolation of vesicles produced in vitro. 
Surprisingly, vesicles are devoid of three different 
Sec proteins required for budding (Secl2p, Sarlp, 
Secl3p) but contain Sec proteins that are required 
for targeting (Sec22p, Yptlp). 
Proteins that are required for vesicle budding 
have been purified by several strategies. A complex 
that includes two Sec proteins (Sec23p and Sec24p) 
has been purified using biochemical complementa- 
tion of a sec23 mutant lysate as a functional assay. 
The small GTP-binding protein Sarlp has been 
shown to be removed selectively from the cytosol 
fraction in cells that overproduce the integral mem- 
brane glycoprotein Secl2p, which is also required 
for vesicle budding. Complementation of such a de- 
pleted cytosol fraction provides a functional assay 
that has allowed purification of Sarlp. Because the 
budding reaction requires intact membranes, it has 
not been possible to isolate a functional form of 
Secl2p from detergent-solubilized membranes. 
However, deletion of the carboxyl-terminal luminal 
domain and the single membrane anchor segment of 
Secl2p releases an amino-terminal cytosolic do- 
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