624
Biogenesis, Structure and Function of Lysosomes
COPI coats also recognize both tyrosine-
based and dileucine motifs. An additional
family of proteins called Golgi-localizing,
gamma adaptin ear homology domain,
ARF-binding (GGAs) form vesicular coats
and bind to dileucine motifs in the M6PRs.
The GGAs are thought to participate in
lysosomal biogenesis and/or traf±cking of
the M6PRs.
Rabs and SNAREs are also required
for this vesicular transport. Rab7 is most
important for the targeting of lysosomal
enzymes
to
lysosomes
while
Rab9
is
important for targeting the M6PRs from
endosomes to the Golgi.
3
Biogenesis of the Yeast Vacuole
We have a more complete understanding
of protein targeting to the yeast vacuole
compared to the biogenesis of mammalian
lysosomes in part because of the power of
yeast genetics. The initial stages of protein
synthesis and transport through the ER
and
Golgi,
glycosylation
of
lysosomal
enzymes,
packaging
the
enzymes
into
vesicles, and docking and fusion of those
vesicles are similar to processes described
for mammalian cells.
3.1
Proteins in the Lumen
Targeting of vacuolar lumen proteins does
not require M6P or any other glycosy-
lation signal. The vacuolar targeting of
carboxypeptidase Y (CPY) has been stud-
ied in detail. More than 50 vacuolar protein
sorting (
Vps
) gene product mutations re-
sult in CPY missorting and abnormal
vacuolar morphology. The proper sorting
of CPY requires a linear peptide sequence
in the amino-terminal propeptide of the
protein. A CPY receptor, Vps10p, binds to
this region of CPY in the Golgi and the
complex traf±cks to a prevacuole compart-
ment called a
multivesicular body
(MVB)
prior to entering the vacuole. The Vps10p
recycles to the Golgi. Various t-SNAREs
and v-SNAREs are required for vesicle fu-
sion with the MVBs or for fusion of MVBs
with the vacuole (Fig. 3).
Two vacuolar proteins, aminopeptidase
I(API)and
α
-mannosidase, enter the vac-
uole independent of any
VPS
genes. They
require gene products from the cytoplasm-
to-vacuole targeting (Cvt) pathway, which
has considerable overlap with genes re-
quired for macroautophagy (Fig. 3). Both
pathways utilize double-membrane vesi-
cles to initially sequester material. The
Cvt pathway selectively sequesters API or
α
-mannosidase multimers while macroau-
tophagy isolates cytoplasm including API
and
α
-mannosidase. Macroautophagy is
stimulated by carbon or nitrogen starva-
tion and is discussed in Sect. 5.3.
3.2
Proteins in the Membrane
Another vacuolar protein sorting pathway
for vacuolar membrane proteins bypasses
the MVBs and targets Golgi-derived vesi-
cles directly to the vacuole. Two proteins
that are targeted to the vacuole as mem-
brane proteins, alkaline phosphatase (ALP)
and the t-SNARE, Vam3p, follow this path-
way to the vacuole (Fig. 3). The yeast
homolog of AP-3 coats the vesicles con-
taining these vacuolar proteins, and the
AP-3 appears to be recruited by tyrosine
or dileucine peptide sequences in the cy-
toplasmic tails of ALP and Vam3p. Many
VPS
genes are common for the CPY and
ALP pathways, but mutants in speci±c
VPS
genes selectively disrupt one pathway or
the other.
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