Intracellular Transport of Proteins 
Luminal Hsc70 may exert its influence through 
interaction with the integral membrane complex 
of Sec proteins. Sec63p, which is a member of 
this complex, has a luminal domain with se- 
quence homology to a bacterial heat-shock pro- 
tein known to interact with the Escherichia coli 
homologue of Hsc70. Luminal Hsc70 may con- 
trol the assembly and disassembly of the translo- 
cation complex by binding to Sec63p. 
Vesicle Transport Early in the 
Secretory Pathway 
Subsequent stages in the secretory pathway in- 
volve protein sorting and transport from the ER to 
the Golgi apparatus and from there to the cell 
surface. Genes required for each of these steps 
are being evaluated by molecular cloning and by 
development of cell-free reactions that measure 
individual steps in the transport process. An assay 
that depends on Sec proteins has been reconsti- 
tuted in vitro. Yeast a-factor precursor is translo- 
cated into the ER lumen of gently lysed yeast 
spheroplasts. In the presence of soluble proteins 
and ATP, the precursor is transferred to the Golgi 
apparatus. This system allows the purification 
and functional characterization of Sec proteins. 
Transfer of secretory proteins from the ER to 
the Golgi apparatus is mediated by small vesicle 
carriers. There are two categories of sec mutants 
defective in this limb of the pathway: mutant 
cells that accumulate ER tubules at a restrictive 
temperature (class I; sec 12, -13, -16, and -23) 
and mutant cells that also accumulate several 
thousand 60-nm vesicles (class II; sec 17, -18, 
and -22). Genetic epistasis tests indicate that 
class I genes must execute their function prior to 
class II genes. This implies that class I gene prod- 
ucts participate in the production of the 60-nm 
vesicles that are consumed, by fusion with the 
Golgi apparatus, through the action of class II 
gene products. Genetic interactions among 
members of class I genes and of class II genes 
suggest that the Sec proteins in each group act in 
a complex, or at least in a concerted manner, to 
perform their respective roles in vesicle budding 
or fusion. 
Transport of a-factor precursor in vitro is me- 
diated by diffusible vesicles. Transport vesicles 
contain core-glycosylated precursor and are phys- 
ically separable from donor ER and target Golgi 
membranes. Budding of vesicles from the ER re- 
quires a crude cytosol fraction, ATP, Secl2p, 
Sec23p, and GTP. Fusion of the vesicles to the 
Golgi compartment is measured by the conver- 
sion of core-glycosylated precursor to a more 
highly glycosylated form. Enriched transport vesi- 
cles target to the Golgi compartment and then 
fuse in distinct subreactions that require cytosol, 
Ca^"^, ATP, and only a subset of Sec proteins. Addi- 
tional proteins in the cytosol fraction that facili- 
tate vesicle budding are being purified. 
Vesicle budding in the secretory pathway was 
thought to be mediated by clathrin, a structure- 
forming protein that spontaneously assembles 
into coats about the size of small transport vesi- 
cles. A test of clathrin function was performed by 
molecular cloning and disruption of the chromo- 
somal genes encoding the large and small sub- 
units of the protein. Clathrin is important but not 
essential for cell growth. Deletion mutations that 
eliminate clathrin produce sickly cells that never- 
theless are competent for most aspects of protein 
transport. A specific lesion in a-factor precursor 
processing, an event that occurs in the Golgi ap- 
paratus, is deficient in clathrin mutant cells. Al- 
ternative structure-forming proteins that serve 
more essential roles in secretion may be discov- 
ered using the Sec protein-dependent vesicle- 
budding reaction. 
382 
