Download A role of SAND-family proteins in endocytosis

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Biochemical Society Transactions (2005) Volume 33, part 4
A role of SAND-family proteins in endocytosis
D. Poteryaev1 and A. Spang
Friedrich Miescher Laboratory of the Max Planck Society, Spemannstrasse 39, Tuebingen D-72076, Germany
Abstract
Caenorhabditis elegans has recently been used as an attractive model system to gain insight into
mechanisms of endocytosis in multicellular organisms. A combination of forward and reverse genetics
has identified a number of new membrane trafficking factors. Most of them have mammalian homologues
which function in the same transport events. We describe a novel C. elegans gene sand-1, whose loss
of function causes profound endocytic defects in many tissues. SAND-1 belongs to a conserved family of
proteins present in all eukaryotic species, whose genome is sequenced. However, SAND family has not been
previously characterized in metazoa. Our comparison of C. elegans SAND-1 and its yeast homologue, Mon1p,
showed a conserved role of the SAND-family proteins in late steps of endocytic transport.
Identification of new endocytosis genes
in multicellular organisms
Endocytosis is a key process conserved in all eukaryotes. It
is essential for the uptake of extracellular nutrients and the
regulation of membrane dynamics. Furthermore, endocytosis
participates in the cell’s reaction to extracellular stimuli by
desensitizing, down-regulating or recycling receptors and
membrane proteins [1]. Genetic screens for effectors of endocytosis have been carried out successfully in the yeast Saccharomyces cerevisiae, yielding a number of genes which
participate in this process [1]. Many of them have homologues
in multicellular organisms, whose functions are similar to or
identical with their yeast counterparts. However, the greater
complexity of higher eukaryotes, compared with yeast, suggests the existence of many variations in endocytic transport
in different cells and tissues. This can be exemplified by the
Ypt/Rab family of small GTPases. The yeast genome contains
11 members of the Ypt family, nine of which were shown to
participate in different steps of vesicular transport pathways,
including endocytosis. The human Rab family comprises
about 60 members, of which only ten are ubiquitous and
have clear functional homologues in yeast. The other Rabs
are believed to be required for either specialized cell functions
or only at certain developmental stages [2].
Forward genetics in higher eukaryotes has been most
successful in the two model organisms Drosophila melanogaster and Caenorhabditis elegans. Over the past few years,
two screens for endocytosis-defective mutants in C. elegans
have been performed [3]. The screens employed two
endocytic models: receptor-mediated endocytosis of yolk
Key words: Caenorhabditis elegans, endocytosis, Rab7, receptor-mediated endocytosis defective
(rme) screen, SAND family, vacuole.
Abbreviations used: CPY, carboxypeptidase Y; cup, coelomocyte uptake defective; GFP,
green fluorescent protein; HOPS, homotypic fusion and vacuolar protein sorting complex;
MLIV, mucolipidosis type IV; rme, receptor-mediated endocytosis defective; SNARE, soluble Nethylmaleimide-sensitive fusion protein attachment protein receptor; ssGFP, secreted soluble
GFP.
1
To whom correspondence should be addressed (email dmitry.poteryaev@tuebingen.
mpg.de).
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in oocytes [rme (receptor-mediated endocytosis defective)
screen] and fluid-phase endocytosis by specialized scavenger
cells residing in the worm’s body cavity, called coelomocytes
[cup (coelomocyte uptake defective) screen]. Yolk uptake by
maturing oocytes is an example of the receptor-mediated endocytosis pathway in many species. Yolk storage vesicles are
thought to be the oocyte equivalent of late endosomes or lysosomes but with low proteolytic activity. A C. elegans strain
carrying a yolk protein YP170 fusion to GFP (YP170–GFP,
where GFP stands for green fluorescent protein) has been
a useful tool to study receptor-mediated endocytosis and
helped to characterize a number of mutants affecting this
pathway [4]. Among the novel conserved genes identified in
the rme screen were rme-1 and rme-8 (Figure 1). RME-1
is a member of a conserved class of EH (Eps15-homology)-domain proteins and is associated with the periphery
of endocytic organelles, suggesting a direct role in endocytic
transport. Evidence from studies of nematode and mammalian RME-1 established a function for RME-1 in recycling, specifically in the exit of membrane proteins from
recycling endosomes. These studies have shown a conserved
function in endocytic recycling for the RME-1 family of EH
proteins [5]. Another gene, rme-8, whose loss of function
causes the RME phenotype in C. elegans [6], has been later
studied in Drosophila. Analysis of Drosophila Rme-8 mutants
showed that internalization of receptors and the uptake
of tracers were blocked. Rme-8 has been then shown to
participate in clathrin uncoating, by interaction with Hsc704 through its J-domain. Thus Rme-8 appears to function
as an unexpected but critical co-chaperone with Hsc70 in
endocytosis [7]. The C. elegans cup screen was based upon
monitoring the uptake of ssGFP (secreted soluble GFP)
from the body cavity by coelomocytes. The most remarkable
gene identified so far in the cup screen is cup-5. Although
cup-5 mutants are not defective in the initial uptake, the
endocytic tracers like ssGFP are not degraded in lysosomes.
Instead, they accumulate in large vacuoles that are positive
for both late endosomal and lysosomal markers. CUP-5 is
the C. elegans functional orthologue of human mucolipin-1
Localization and Activation of Ras-like GTPases
Figure 1 A model of endocytic traffic and functions of endocytic
effectors discovered in C. elegans
RAB-5, RAB-11 and RAB-7 participate in early endosome (EE) formation,
recycling from endocytic recycling compartment and EE to late endosome (LE) transport respectively. The major endocytic traffic route to
lysosomes from plasma membrane-coated pits is shown, incorporating
present views [9] of lysosome–LE fusion and re-formation of lysosomes
from the resulting hybrid organelles. CUP-5 might control the process of
lysosome re-formation by regulating the calcium flux. SAND-1 is thought
to function at the EE to LE step. CCV, clathrin-coated vesicle.
[8]. Mutations in mucolipin-1 cause MLIV (mucolipidosis
type IV), a disease characterized by psychomotor retardation
and ophthalmological abnormalities. In MLIV patients, cells
of several tissues accumulate large vacuoles that are presumed
to be abnormal lysosomes. CUP-5 is a calcium channel and
functions in the biogenesis of lysosomes originating from the
so-called hybrid organelles (Figure 1) [8,9]. Late endosomes
dock and fuse with lysosomes in a SNARE (soluble Nethylmaleimide-sensitive fusion protein attachment protein
receptor)-dependent reaction that requires the efflux of
calcium from lysosomes or endosomes. The fusion might
occur either in a restricted manner (kiss-and-run) or in full,
resulting in an endosome–lysosome hybrid organelle. According to the present view, late-endosome components are
sorted into discrete subdomains and separated from hybrid
organelles to enable the re-formation of lysosomes and the
retrieval of late-endosome components. Calcium- and pHdependent condensation of contents facilitates lysosome reformation [9].
Remarkably, most of the genes identified in these studies,
while having functional homologues in vertebrates, are not
present in yeast. This stresses the importance of using metazoan model organisms, such as C. elegans, for which powerful
genetic methods are available, in deciphering endocytosis.
Clearly, there are many unanswered questions, and new
effectors of endocytosis are awaiting discovery.
SAND family and endocytosis
The C. elegans mutant allele or552 was identified in a screen
for temperature-sensitive embryonic lethal mutations [15].
Detailed analysis of the or552 phenotype led us to speculate
that the affected gene is involved in late steps of endocytic
transport and probably acts on the same level as rab-7. We
found that or552 is a nonsense mutation in a gene that
belongs to the SAND family. According to the C. elegans
nomenclature it was named sand-1. SAND was first identified
as an open reading frame in the yeast S. cerevisiae genome.
An orthologous gene was found later in Fugu rubripes and
was called SAND. By now, it is clear that SAND genes are
present in every major eukaryotic taxon. Fungi, plasmodia,
slime mould, nematodes, sea squirt and plants all have one
SAND protein encoded in their genome. Interestingly, in
vertebrate species, including humans, two SAND genes are
always present. Sequence analysis of the SAND family has
failed to detect any significant similarities with known protein
domains. Therefore the SAND proteins are ancient and
belong to an absolutely distinct protein family. The most
conspicuous feature of sand-1 (or552) mutants is the presence
of large intracellular membrane-bounded granules in the early
embryo. These granules are a result of defective endocytic
transport of yolk protein in maturing oocytes (Figure 2A).
The fact that at permissive temperature the mutant animals
survive, although still having defects in endocytosis, allowed
us to investigate the role of sand-1 also in somatic tissues. In
coelomocytes we observed a similar defect: accumulation of
extremely large vacuoles of endocytic origin (Figure 2B).
The only member of the SAND family whose biological
function has been investigated is the budding yeast gene
MON1 [10]. Mon1p is involved in autophagy, vacuolar morphology, cytoplasm-to-vacuole trafficking, and is required
for homotypic vacuole fusion. In other words, Mon1p is
required in nearly all membrane trafficking pathways, where
the vacuole represents the terminal acceptor compartment.
Biochemical experiments in budding yeast have identified
four distinct stages in homotypic vacuole fusion: priming
of the vacuoles, where cis-SNARE complex disassembles and
the C-Vps/HOPS (homotypic fusion and vacuolar protein
sorting complex) is released from the SNAREs; tethering,
which requires the interaction of the C-Vps/HOPS complex
with the rab GTPase Ypt7, and, finally, docking and fusion.
Mon1p forms a stable complex with another protein, Ccz1p.
This complex is required for homotypic vacuole fusion at
tethering/docking stage; however, only Mon1p plays a direct
role, as determined by antibody inhibition assays [11]. The
complex of Ccz1p and Mon1p is essential for SNARE pair
association during homotypic vacuole fusion and, probably,
in other trafficking pathways to the vacuole [11,12]. Apparently, Ypt7p, Mon1p and Ccz1p act on the same level.
The deletion phenotypes of these genes are highly similar,
regarding the vacuole integrity and membrane transport.
Moreover, overexpression of YPT7 completely suppressed
the effect of a CCZ1 deletion [13]. Whether Ypt7p and
either Mon1p or Ccz1p interact physically remains an open
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Biochemical Society Transactions (2005) Volume 33, part 4
Figure 2 Conserved role of sand-1/MON1 in endocytosis
(A) Abnormal receptor-mediated endocytosis in sand-1 (or552) mutants. Cytoplasm of an early embryo contains enlarged
yolk granules. (B) Coelomocytes in sand-1 (or552) accumulate large vacuoles of endocytic origin. Uptake of BSA–Texas Red
injected into the body cavity is shown. (C) Expression of C. elegans sand-1 in a yeast strain deficient for MON1 can restore the
vacuole targeting. CPY maturation assay is shown. Arrows indicate: p1, CPY precursor in ER; p2, glycosylated CPY precursor
in Golgi or pre-vacuolar compartment; m, mature form in the vacuole. The time of chase after the pulse is shown in min.
question. Kucharczyk et al. [13] reported the interaction of
Ccz1p with Ypt7p, whereas Wang et al. [11,12] were not
able to reproduce these results. They also failed to detect an
interaction between Mon1p and Ypt7p [11,12]. Still, it may
be possible that these interactions are transient, whereas the
Ccz1p–Mon1p complex is very abundant and stable. It is
therefore tempting to suggest that Ccz1p and Mon1p are
part of an Ypt7p effector complex. It should be noted that
Ccz1p has not been found in genomes of any other eukaryotic
species. Therefore this association is not evolutionarily conserved. Moreover, it is conceivable that in distant eukaryotic
taxons, SAND-interacting proteins evolved independently
and are different. To date, only one metazoan SAND has been
experimentally studied. Human HSRG1 has been identified
as a protein whose expression is induced at high levels in fibroblasts by HSV-1 (herpes simplex virus 1) infection [14].
Although these experiments did not go any further, one is
tempted to speculate about an involvement of HSRG1 in
endocytosis and a possible link of endocytic transport and
HSV-1 infection.
We propose that sand-1 may be a functional orthologue
of yeast MON1. To test this hypothesis, we tried to complement the protein transport defect of ∆mon1. CPY
(carboxypeptidase Y) is transported to the vacuole through
the endoplasmic reticulum, Golgi complex and endosome.
Because CPY is post-translationally modified, its transport
through the secretory pathway is easily followed. In the wildtype strain, CPY matured with a half-time of 5–10 min. In
∆mon1, the majority of the protein was found in the precursor
forms even after 40 min of chase, which is in agreement with
previous findings [11]. The delay in CPY maturation resulted
from a defective cargo transport from the pre-vacuolar compartment to the vacuole. The ∆mon1 strain transformed with
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sand-1 showed a marked increase in the kinetics of CPY
maturation. After a 30-min chase, more than half of the
CPY was in the mature form (i.e. reached the vacuole) (Figure 2C). Thus overexpression of sand-1 can at least partially
rescue the function of its yeast orthologue. We have shown
here that the C. elegans SAND-1 is required for late steps of
endocytosis in oocytes, embryos, coelomocytes and, possibly,
in many other tissues. Furthermore, the C. elegans SAND-1
is the functional orthologue of the S. cerevisiae Mon1p, suggesting a conserved role of these proteins in different systems.
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Received 9 March 2005