Intracellular Protein Transport 
may be mediated by an independent organelle, 
the peroxisome. Mutations that block peroxiso- 
mal assembly also block FBPase degradation. Fur- 
thermore, a peroxisomal enzyme, acyl-CoA thio- 
lase, is subject to the same glucose-stimulated, 
vacuolar-dependent degradation process. One 
possible explanation of these results is that 
FBPase may be imported into or somehow asso- 
ciated with peroxisomes and then localized to 
the vacuole by autophagic uptake of the 
peroxisome. 
Vesicle Transport Early in the Secretory 
Pathway 
Subsequent stages in the secretory pathway in- 
volve protein sorting and transport from the en- 
doplasmic reticulum to the Golgi apparatus and 
from there to the cell surface. Genes required for 
each of these steps are being evaluated by molec- 
ular cloning and by development of cell-free reac- 
tions that measure individual steps in the trans- 
port process. An assay that depends on Sec 
proteins has been reconstituted in vitro. Yeast 
a-factor precursor is translocated into the ER lu- 
men of gently lysed yeast spheroplasts. In the 
presence of soluble proteins and ATP, the precur- 
sor is transferred to the Golgi apparatus. This sys- 
tem allows the purification and functional charac- 
terization of Sec proteins. 
Transfer of secretory proteins from the ER to 
the Golgi apparatus is mediated by small vesicle 
carriers. In this limb of the pathway, defective sec 
mutants fall into two categories: class I, mutant 
cells that accumulate ER tubules at a restrictive 
temperature {SEC 12, -13, -16, and -23); and 
class II, mutant cells that also accumulate several 
thousand 60-nm vesicles (SEC17, -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 products partici- 
pate in the production of the 60-nm vesicles that 
are consumed, by fusion with the Golgi appara- 
tus, through the action of class II gene products. 
Genetic interactions among members of class I 
and 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 vesi- 
cle budding or fusion. 
Transport of the a-factor precursor in vitro is 
mediated by diffusible vesicles. Transport vesi- 
cles contain a core-glycosylated precursor and 
are physically separable from donor ER and target 
Golgi membranes. Budding of vesicles from the 
ER requires a crude cytosol fraction, ATP, 
Secl2p, Secl3p, Sec23p, GTP, and a ras-like 
GTP-binding protein, Sarlp. Fusion of the vesi- 
cles to the Golgi compartment is measured by 
conversion of the precursor to a more highly gly- 
cosylated form. Enriched transport vesicles target 
to the Golgi compartment and then fuse in dis- 
tinct subreactions that require cytosol, Ca^"^, ATP, 
and only a subset of Sec proteins. 
To allow the purification of functional Sec23p, 
we have developed an assay based on restoration 
of transport in sec23 lysates by wild-type protein 
fractions. The purified protein has been isolated 
in two forms: a Sec23p monomer, and a hetero- 
oligomer that consists of Sec23p and a 105-kDa 
protein (Sec24p) that is also required for vesicle 
budding. The SEC 2 3 and -24 genes have both 
been cloned, and though the sequences confirm 
the observed molecular weights of the isolated 
polypeptides, no homology to known proteins 
was found. 
Localization of Sec proteins in yeast has met 
with only limited success. Yeast morphologic 
analysis is limited by the small cell size and the 
difficulty of specimen preservation. Fortunately 
the functional and structural conservation of Sec 
proteins allows their localization in mammalian 
cells. The first and most favorable example is 
Sec23p, where antibodies directed against the 
yeast protein cross-react with the mammalian ho- 
mologue. Immunolocalization studies reveal a 
striking enrichment of Sec23p in the cytoplasmic 
pocket that separates the transitional ER and the 
cis face of the Golgi complex. This location is 
completely consistent with the role proposed for 
yeast Sec23p and further confirms that the secre- 
tory pathway is fundamentally conserved across 
the broad spectrum of the eukaryotic kingdom. 
354 
