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Address correspondence to Randy Schekman, Dept. of Molecular and Cell Biology, Howard Hughes Medical Institute, University of California, Berkeley, CA 94720. Tel.: (510) 642-5686. Fax: (510) 642-7846. E-mail: schekman{at}UCLink4.berkeley.edu
Exocytic vesicles that accumulate in a
temperature-sensitive sec6 mutant at a restrictive temperature
can be separated into at least two populations with different buoyant
densities and unique cargo molecules. Using a sec6 mutant
background to isolate vesicles, we have found that vacuolar protein
sorting mutants that block an endosome-mediated route to the vacuole,
including vps1, pep12, vps4, and a
temperature-sensitive clathrin mutant, missort cargo normally
transported by dense exocytic vesicles, such as invertase, into light
exocytic vesicles, whereas transport of cargo specific to the light
exocytic vesicles appears unaffected. Immunoisolation experiments
confirm that missorting, rather than a changed property of the normally
dense vesicles, is responsible for the altered density gradient
fractionation profile. The vps41
and apl6
mutants, which block transport of only the subset of vacuolar proteins
that bypasses endosomes, sort exocytic cargo normally. Furthermore, a
vps10 sec6 mutant, which lacks the sorting
receptor for carboxypeptidase Y (CPY), accumulates both invertase and
CPY in dense vesicles. These results suggest that at least one branch of
the yeast exocytic pathway transits through endosomes before reaching
the cell surface. Consistent with this possibility, we show that
immunoisolated clathrin-coated vesicles contain
invertase.
Key Words: protein sorting; TGN; endosome; VPS; yeast secretion
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