Vitellin cleavage products are proteolytically degradedby ubiquitination in stick insect embryos

Antonella Cecchettinia, Maria Teresa Loccib, Massimo Masettic, Anna Maria Faustod,Gabriella Gambellinid, Massimo Mazzinid, Franco Giorgia,*

aDepartment of Human Morphology and Applied Biology, University of Pisa, Via A, Volta 4, Pisa 56126, ItalybDepartment of Experimental Pathology, University of Pisa, Pisa, Italy

cDepartment of Ethology Ecology and Evolution, University of Pisa, Pisa, ItalydDepartment of Environmental Sciences, Tuscia University, Viterbo, Italy

Received 31 May 2002; revised 18 November 2002; accepted 21 November 2002

Abstract

Vitellin polypeptides are proteolytically processed in ovarian follicles and embryos of the stick insect Carausius morosus. Data show that

vitellin polypeptide A3 of 54 kDa is processed to yield polypeptide A3p of about 48 kDa upon completion of ovarian development, whereas

vitellin polypeptide A2 of 90 kDa yields polypeptide E9 during embryonic development. As vitellin polypeptides are processed, polypeptides

A3p and E9 are transferred from the yolk granules to the cytosolic space of the vitellophages and start to express a ubiquitin reactivity. At the

confocal microscope, anti-ubiquitin antibodies label specifically numerous small yolk granules and the cytosolic space of vitellophages.

During embryonic development, ubiquitin carrying granules undergo acidification in much the same way as larger yolk granules. However,

only these latter organelles are capable of converting a latent cysteine pro-protease into an active yolk protease upon acidification of their

luminal space. These data are interpreted as indicating that ubiquitin-like polypeptides are restricted to small granules throughout ovarian and

embryonic development, and that vitellin cleavage products are ubiquitinated following acidification of large yolk granules and transfer to

the cytosolic space of the vitellophages.

q 2003 Elsevier Science Ltd. All rights reserved.

Keywords: Carausius morosus; Embryos; Ubiquitin; Vitellin polypeptides

1. Introduction

In eukaryotic cells, proteins targeted to the lysosomal

pathway for degradation are taken up endocytically from the

extracellular milieu (Gruenberg and Maxfield, 1995; Futter

et al., 1996) or confined to such selected areas of the

cytoplasm as autophagosomes or cytolysosomes (Klionsky

and Ohsumi, 1999). In either case, confinement of active

proteolytic enzymes to membrane-bound organelles helps

maintaining the degradative processes physiologically

separated from other metabolic activities of the cell

(Baumeister et al., 1998).

In oviparous species, vitellin polypeptides provide stores

of nutritional substances to sustain embryonic development

metabolically (Kunkel and Nordin, 1985; Giorgi et al.,

1999). To this end, they are partitioned in endosome-like

organelles, the yolk granules (Opresko et al., 1980; Giorgi

et al., 1999), by the ability of the growing oocyte to undergo

receptor mediated endocytosis (Raikhel and Dhadialla,

1992; Sappington et al., 1995; Snigirevskaya et al., 1997).

Due to their endocytic origin via membrane-bound

organelles, vitellin polypeptides have long assumed to be

degraded proteolytically along the lysosomal pathway

(Yamashita and Indrasith, 1988; Nordin et al., 1990). In

line with this assumption, several lysosomal, maternally

encoded, pro-proteases were found in developing embryos

(Indrasith et al., 1988; Cho et al., 1991a,b; Takahashi et al.,

1993; Cho et al., 1999) and shown to be associated with yolk

granules together with vitellin polypeptides (Takahashi

et al., 1996; Giorgi et al., 1997). With the initiation of

embryonic development, these latent pro-proteases are

gradually converted to active proteolytic enzymes by

acidification of the yolk granules (Fagotto, 1991; Nordin

0968-4328/03/$ - see front matter q 2003 Elsevier Science Ltd. All rights reserved.

doi:10.1016/S0968-4328(02)00057-4

Micron 34 (2003) 39–48

www.elsevier.com/locate/micron

* Corresponding author. Tel.: þ39-50-501078; fax: þ39-50-501085.

E-mail address: giorgif@biomed.unipi.it (G. Franco).

et al., 1991; Mallya et al., 1992; Fausto et al., 2001), causing

vitellin polypeptides to undergo limited proteolysis and to

yield a number of lower molecular weight vitellin cleavage

products (Masetti and Giorgi, 1989; Masetti et al., 1998).

However, it is presently unknown whether these maternally

encoded pro-proteases render the embryonic yolk granules

proteolytically autonomous, that is to say capable of

degrading all vitellin polypeptides exhaustively.

A large body of evidence has by now indicated that many

structural and regulatory proteins in eukaryotic cells are

degraded by the 26S proteasome via covalent attachment of

multiple ubiquitin molecules (Haire et al., 1995; Hershko

and Ciechanover, 1998). Besides affecting protein turn-

over, ubiquitination is also known to control protein

confinement within cell compartments by helping them to

translocate across the enclosing membrane (Mattack et al.,

1998; Wilkinson, 2000). For instance, many secretory

proteins are degraded in the cytosol by the ubiquitin-

dependent pathway, even though originally confined within

the lumen of endoplasmic reticulum cisternae (Hiller et al.,

1996; Bonifacino and Weisman, 1998). Similarly, several

trans-membranous receptors can be removed from the

plasma membrane and targeted to the lysosomal pathway by

ubiquitin attachment (Hicke, 1999; Dunn and Hicke, 2001).

Both these examples demonstrate the existence of a

complex interplay between the lysosomal and the ubiqui-

tin-dependent pathways in eukaryotic cells (Hofman and

Falquet, 2001).

We have recently shown that several anti-vitellin

reactivities appear in vitellophages of late embryos of the

stick insect Carausius morosus. This indicates that vitellin

cleavage products may escape from the yolk granules and

gain access to the surrounding cytoplasm (Cecchettini et al.,

2001; Fausto et al., 2001; Cecchettini et al., 2002). In view

of the complex networks connecting the lysosomal and

ubiquitin-dependent pathways, we thought it feasible for the

insect vitellin cleavage products to be degraded proteoly-

tically in cell sites external to the yolk granule compartment.

The present study aims at exploring this possibility and to

verify whether yolk utilization along the lysosomal pathway

may entail completion of vitellin degradation via the

ubiquitin-dependent pathway.

2. Material and methods

2.1. Rearing and sample preparations

Specimens of the stick insect C. morosus (Br.) (Phasma-

todea, Lonchodinae) were reared on English ivy leaves and

maintained in aerated cages at constant temperature and

humidity. Newly laid eggs were collected weekly, main-

tained at the same conditions throughout development.

Embryos were staged according to Fournier (1967) upon

dissection. Vitellophages were isolated from yolk sacs

freshly dissected in phosphate buffered saline (PBS) from

stage V–VII embryos and soon processed for both light and

electron microscope observations. Protein concentration of

tissue extracts was determined spectrophotometrically using

the procedure of Bradford (1976).

2.2. Scanning electron microscopy

Vitellophages or intact yolk sacs were newly dissected in

PBS from stage V–VII embryos and fixed in Karnovsky’s

fixative as previously reported (Fausto et al., 1994). They

were then thoroughly rinsed in 0.1 M cacodylate buffer at

pH 7.2, post-fixed for two additional hours in 1% osmium

tetroxide in 0.1 M cacodylate buffer at pH 7.2, dehydrated in

alcohol and eventually critical point dried in a Balzer’s

apparatus (Balzers Union AG, Liechtenstein) equipped with

liquid CO2. Samples were subsequently mounted on

aluminium stubs, manually fractured and metal shadowed

in a sputtering unit under a continuous Argon flow. Samples

were observed in a Jeol JSM 5024 scanning electron

microscope (Jeol, Akishima, Tokyo, Japan).

2.3. Monoclonal and polyclonal antibodies

Monoclonal antibodies (mAb) against vitellin polypep-

tides were raised in BALB/c mice injected subcutaneously

with 100 ml of tissue homogenates freshly prepared from

either newly dissected ovarian follicles or embryonic yolk

sacs as previously reported (Masetti et al., 1998). Antibody

producing lymphocytes were extracted from the spleen of

the injected mice and fused with mouse myeloma cell line

P3X63-Ag8.653. Hybridoma cell lines were selected in

HAT medium and screened by ELISA and western blotting

(Kohler and Milstein, 1975). For this study, mAbs A1C3

and 1B12 were selected and shown to react with vitellin

polypeptides A2 and A3 of C. morosus, respectively. A

polyclonal antibody (pAb) anti-vitellin antiserum was raised

in white New Zealand rabbits using protein homogenates

from newly laid eggs of C. morosus. A mouse anti-pro-

protease pAb was originally developed by Prof J.H. Nordin

(University of Massachusetts, USA) in Blattella germanica

(Liu et al., 1997; Liu and Nordin, 1998) and recently proved

to be cross-reactive with vitellin polypeptide B2 in the stick

insect C. morosus (Fausto et al., 2001). Monoclonal and

polyclonal antibodies against ubiquitin were purchased

from Sigma (Sigma Chemicals, St Louis, MO, USA) and

used according to the protocol recommended by the

manufacturer. Their specificity was checked by western

blots against known aliquots of purified ubiquitin.

2.4. Polyacrylamide gel electrophoresis

and western blotting

Samples of early (Ev1 and Ev2) and late (Lv) ovarian

follicles, chorionated oocytes (CH), newly laid eggs (E) and

embryos at different developmental stages (II1–VII5) were

homogenized in 250 ml of 50 mM Tris–HCl buffer at pH

A. Cecchettini et al. / Micron 34 (2003) 39–4840

6.8 containing a mixture of proteolytic inhibitors (1 mM

PMSF, 1.4 mM pepstatin, 1.2 mM cystatin, 3 mM aprotinin,

10 mM iodoacetic acid and 5 mM EDTA). Following

spinning at 10,000 rpm in a refrigerated bench centrifuge,

samples were diluted with an equal volume of 50 mM Tris–

HCl buffer at pH 6.8 containing 5% b-mercaptoethanol, 5%

sodium dodecyl sulfate (SDS) and boiled for 3 min. Gel

electrophoresis was carried out according to Laemmli

(1970) using a 5–15% polyacrylamide gradient and run

overnight in a Bio-rad Protein II apparatus (Bio-Rad

Laboratories, Hercules, CA, USA) at 15 8C with 0.1%

SDS in the upper electrode reservoir. Polyacrylamide gels

were stained for 1 h in 0.1% Coomassie Brilliant Blue R-

250 in 30% acetic acid–10% methanol, destained in the

same solution and eventually dried under vacuum at 60 8C.

Molecular weights of vitellin polypeptides were estimated

as previously reported (Giorgi et al., 1993), using Bio-rad

standards and plotting the distance migrated by the resolved

protein fractions against log of their respective molecular

weight. Electrophoresed polyacrylamide gels were placed

onto nitro-cellulose sheets and trans-blotted for 2 h at 16 8C

following the procedure of Towbin et al. (1979). These

nitro-cellulose sheets were subsequently allowed to react

with primary mouse antibodies for 3 h at room temperature

and eventually treated for an additional hour with goat anti-

mouse secondary antibodies conjugated with alkaline

phosphatase (Bio-Rad Laboratories, Hercules, CA, USA).

Reactivity was revealed by incubation with Bio-rad

developers and blocked by addition of 0.1 M HCl.

2.5. Scanning laser confocal microscopy

Embryos from V to VII developmental stages of C.

morosus were fixed in 4% formaldehyde for 4 h in PBS,

thoroughly rinsed in the same buffer and allowed to infiltrate

for 3 days in a 30% solution of sucrose at 4 8C. They were

then embedded in a tissue resin and frozen at 220 8C.

Cryosections of about 20–25 mm in thickness were

prepared and processed for immunocytochemistry follow-

ing standard procedures. Accordingly, they were incubated

overnight at 4 8C with mAbs and eventually treated with

fluorescein conjugated goat anti-mouse secondary anti-

bodies. Slides were then observed in a fluorescent

microscope connected to a TCS 4D confocal scanning

system (Leica Microsystems, Heidelberg, Germany)

equipped with Ar/Kr laser. Fluorescent signals were

detected at 488/568 and 520/590 nm emission wavelengths.

Pictures were downloaded on a Power McIntosh computer

(Apple Computers, Cupertino, CA, USA) and elaborated

using the Photoshop program (Adobe Systems, San Jose,

CA, USA).

2.6. Acidification and cysteine protease activity

Vitellophages freshly dissected from developmentally

different embryos of C. morosus were placed in sterile

Grace’s medium containing 100 nM of the acidotropic

probe Lysotracker (Molecular Probes, OR, USA). Speci-

ficity of Lysotracker uptake was checked by incubation with

bafilomycin (at concentrations ranging from 100 nM to

1.5 mM). Some samples were also investigated for the

presence of cysteine pro-protease by incubation in a mouse

anti-cysteine pro-protease antiserum following permeabili-

zation in 0.5% Triton X-100 in PBS. In another series of

experiments, vitellophages were double labeled for the

simultaneous detection of cysteine pro-protease and yolk

granule acidification.

3. Results

Fig. 1 shows the entire developmental profile of vitellin

polypeptides during both ovarian and embryonic develop-

ment of the stick insect C. morosus. Polypeptides B1, A1, A2

and B2 remain almost invariant from early vitellogenesis up

to stage III of embryonic development, whereas polypeptide

A3 disappears prior to completion of chorionogenesis. At

this ovarian stage, polypeptide A3 of 54 kDa is processed to

a lower molecular weight product to yield polypeptide A3p of

about 48 kDa (Fig. 1). Following stage III of embryonic

development, all major vitellin polypeptides undergo

limited proteolysis, to generate a number of vitellin

cleavage products of lower molecular weights (see blot

against a pAb anti-Vt of Fig. 2). Among these, polypeptide

A2 of 90 kDa is gradually reduced in relative concentration

and completely exhausted by the end of stage VII, its

exhaustion being temporally related with the appearance of

other polypeptides of lower molecular weight. To verify the

possibility that some of these lower molecular weight

polypeptides may result from polypeptide A2 processing,

vitellin extracts from stage II–VII embryos were tested by

western blottings against both pAb and mAb. While mAb

Fig. 1. Ovarian follicles (Ev1, Ev2, Lv and CH), newly laid eggs (E) and

embryos of the stick insect C. morosus at different developmental stages

(III3–VII2) were resolved by polyacrylamide gel electrophoresis under

denaturing conditions (SDS–PAGE) (Left panel) and blotted against a

mAb 1B12 specific for vitellin polypeptide A3 (Right panel). Vitellogenin

(Vg) polypeptides A1, A2, A3, B1 and B2 are indicated. Molecular weight

(Mr) of the major Vg polypeptides are expressed in kDa.

A. Cecchettini et al. / Micron 34 (2003) 39–48 41

A1C3 yields a staining pattern mimicking the develop-

mental profile of polypeptide A2, pAb anti-A2 exhibits an

additional cross-reactivity for polypeptide E9 of 80 kDa

(Fig. 2). These observations indicate that polypeptides A2

and E9 bear a precursor–product relationship to each other,

and that the polypeptide A2 epitopes reacting with mAb

A1C3 are lost upon processing.

Having established the extent by which vitellin poly-

peptides are processed in stick insect embryos, we then

wished to study how they become spatially distributed

amongst yolk granules. Newly laid eggs of the stick insect

C. morosus are characterized by a fluid yolk mass gradually

partitioned into a number of yolk granules during embryonic

development (Fausto et al., 1994). Partitioning occurs by

virtue of the phagocytic activity of vitellophages invading

the ooplasm from the egg periphery (Fausto et al., 1997).

Vitellophages were examined by confocal fluorescence

microscopy, following exposure to anti-vitellin polypeptide

antibodies. Figs. 3(A) and (B) show two vitellophages at

different developmental stages exposed to mAb 1B12. The

labeling patterns provided by this antibody indicate that

polypeptide A3 is initally confined to the yolk granules,

while later it is displaced to the cytosolic space of the

vitellophage. The same interpretation holds true for

vitellophages exposed to mAb A1C3 and pAb anti-A2

(Figs. 3(C) and (D)). However, while mAb A1C3 labels

most of the yolk granule volume, anti-A2 pAb appears only

to react with material harboring the cytosolic space

comprised between adjacent yolk granules. Figs. 3(E) and

(F) are enlargements of previous figures to show details of

the labeling patterns provided by these two antibodies on

yolk granules of stick insect vitellophages. To verify

whether labeling may be due to the yolk granule membrane

preventing anti-A2 pAb access to the yolk granule interior,

fluorescent immunostaining was carried out on cryosections

of stick insect yolk sacs. The observation that, under these

conditions, label becomes uniformly dispersed on both the

yolk granules and the cytosolic space around them suggests

that the yolk granule membrane is most likely acting as a

barrier impeding anti-A2 pAb entrance into the yolk granule

Fig. 2. Embryos of the stick insect C. morosus at different developmental stages (II1–VII5) were resolved by polyacrylamide gel electrophoresis under denaturing

conditions (SDS–PAGE) andblottedagainst: (1) apAbanti-Vt raisedagainst totalvitellin extracts; (2) amAbA1C3specific for vitellin polypeptideA2; (3) a pAbanti-

A2 raised against vitellin polypeptideA2. PolypeptidesA1, A2, A3, B1 andB2 areVgpolypeptides stored inovarian follicles. E20, E9E5 andE4 are vitellin (Vt) cleavage

products resulting from Vg proteolytic processing during embryonic development. Molecular weight (Mr) of the major Vg polypeptides are expressed in kDa.

Fig. 3. Laser confocal microscope analysis of vitellophages (A)–(D) from

stick insect embryos at stage VII of development tested against various, anti-

vitellin polypeptide,mAbs, pAbs.MAb1B12 labels a number of yolk granules

inside the vitellophage (A) andmost of the cytosolic space comprised between

adjacent yolk granules (B). MAb A1C3 labels all yolk granules, but not the

cytosolic spaceof vitellophage (C).Anti-A2 pAb labels the cytosolic space, but

not the yolk granules inside the vitellophage (D). Pictures in (E) and (F) are

enlargements of (C) and (D), respectively. Anti-A2 pAb labels both the yolk

granules and the cytosolic spacewhen applied on cryosections of vitellophages

from stage VII embryos (G) and (H). (A) and (B) (Bar, 40 mm); (C) and (D)

(Bar, 32 mm); (E) and (F) (Bar, 8 mm). (G) and (H) (Bar, 18 mm).

A. Cecchettini et al. / Micron 34 (2003) 39–4842

Fig. 4. Ovarian follicles (Lv and CH), newly laid eggs (E) and embryos at different developmental stages (III3–VII2) were resolved by polyacrylamide gel

electrophoresis under denaturing conditions (SDS–PAGE) and blotted against anti-ubiquitin, pAb and mAb. Ubiquitin labeling is associated with A2, E9 and A3p

polypeptides (see arrows). Polypeptides A1, A2, A3 and B1 and B2 are Vg polypeptides present in ovarian follicle and egg extracts. E20, E9 E5 and E4 are vitellin (Vt)

cleavageproducts resulting fromVgproteolyticprocessingduring embryonicdevelopment.Molecularweight (Mr)of themajorVgpolypeptides are expressed inkDa.

Fig. 5. Vitellophages from early stage VII embryos examined by scanning electron microscopy (A) and light microscopy (B) and (D). (C) is a laser confocal

microscope view of an early stage VII vitellophage exposed to an anti-ubiquitin mAb. (E) is an enlargement of the cytosolic space of a stage VII vitellophage

exposed to the same antibody. (F), is a scanning electron microscope view of a stage VII vitellophage showing some fine particulate material on the yolk

granules. (A)–(C) (Bar, 20 mm); (D) (Bar, 10 mm); (E) and (F) (Bar, 5 mm).

A. Cecchettini et al. / Micron 34 (2003) 39–48 43

(Figs. 3(G) and (H)). The conclusion one can draw from

these observations is that, during embryonic development,

polypeptide E9 gains access to the cytosolic space of the

vitellophages by translocation through the yolk granule

membrane.

Vitellin extracts from ovarian and embryonic stages were

also tested for their ability to interact with anti-ubiquitin

antibodies. Both mono- and polyclonal antibodies interact

with polypeptides A2, A3 and E9, besides labeling a cascade

of lower molecular weight products generated from vitellin

polypeptides in stage VI–VII embryos (Fig. 4). However,

anti-ubiquitin mAb reactivity is restricted to polypeptides

A2 and E9 and expressed throughout ovarian and embryonic

development. By contrast, anti-ubiquitin pAb reacts in

addition with polypeptide A3p following processing to

48 kDa during ovarian development. Based on these

observations, we may reasonably conclude that anti-

ubiquitin reactivity in stick insect embryos is expressed by

both vitellin polypeptides A2 and A3 following processing to

lower molecular weight cleavage products as polypeptides

A3p and E9, respectively.

The latter conclusion raises the question whether

membrane translocation of vitellin polypeptides through

the yolk granule membrane and ubiquitination are causally

related events in stick insect embryos. Ubiquitination could

either be a prerequisite for vitellin polypeptides to be

translocated, or alternatively, vitellin polypeptides could

become ubiquitinated upon translocation from the yolk

granules. To provide an experimental answer to these

queries, vitellophages were examined by confocal

microscopy to determine localization of ubiquitin related

polypeptides. Fig. 5(A) shows a typical vitellophage from

an early stage VII yolk sac. At this developmental stage,

yolk granules are tightly packed within the vitellophage

cytoplasm and highly heterogenous in size (Figs. 5(B) and

(D)). When examined by confocal fluorescence microscopy

following exposure to anti-ubiquitin antibodies, vitello-

phages appeared labeled on two specific cell sites: (a) the

cytosolic space comprised between adjacent yolk granules

and (b) a number of small yolk granules of about 2 mm in

mean diameter (Fig. 5(C)). At higher magnification, these

labeling patterns appeared associated with some particulate

material dispersed in the cytosolic space of the vitellophage

or bound to the yolk granule membrane (Fig. 5(E)). Such a

fine particulate material can also be clearly seen by scanning

electron microscopy in yolk granules of vitellophages of

stage VI (not shown) and stage VII embryos (Fig. 5(F)). The

fluorescent patterns resulting from anti-ubiquitin labeling

characterize all vitellophages of stage VI and VII embryos,

regardless of the nature of the antibody employed. From

these data one can reasonably conclude that anti-ubiquitin

reacting polypeptides are associated with both the cytosolic

Fig. 6. Vitellophages from early stage VII embryos examined by laser confocal microscopy following anti-ubiquitin exposure (A) and by scanning electron

microscopy (B). Vitellophages from early stage VII embryos were also exposed simultaneously to: (C) anti-ubiquitin antibodies (green fluorescence) and to the

acidotropic probe, Lysotracker, (red fluorescence) or to (D) anti-cysteine pro-protease antibody (green fluorescence) and to the acidotropic probe, Lysotracker,

(red fluorescence). (Bar, 10 mm).

A. Cecchettini et al. / Micron 34 (2003) 39–4844

space and small yolk granules. Due to their size and position

inside the vitellophage, these small granules can be easily

identified by scanning electron microscopy amongst larger

yolk granules (compare Figs. 6(A) and (B)).

Since yolk granules in stick insect embryos are known to

become differentially acidified during development (Fausto

et al., 2001), we wished to verify whether access to the

cytosolic space is somehow conditioned by acidification of

the ubiquitin carrying granules. Fig. 6(C) shows several

yolk granules from stage VII vitellophages exposed

simultaneously to anti-ubiquitin antibodies and to an

acidotropic probe to test for their luminal pH. As one can

clearly see, the majority of yolk granules in the vitellophage

is already acidified. However, several small granules appear

to retain the original green color for anti-ubiquitin

reactivity, while several yellow spots occur along the

interface between the red and green granules (Fig. 6(C)). A

partially superimposable staining pattern can also be

detected in vitellophages exposed simultaneously to the

acidotropic probe and to antibodies specific for a lysosomal

cysteine pro-protease (Fig. 6(D)). These observations

confirm that ubiquitin reacting polypeptides are predomi-

nantly stored in small granules, while the remaining vitellin

polypeptides are confined to larger yolk granules. While

both yolk granules and ubiquitin carrying granules undergo

acidification, only vitellin polypeptide translocation to the

cytosolic space is apparently mediated by conversion of a

latent cysteine pro-protease to an active yolk protease. Fig. 7

presents a schematic model of vitellin protein processing in

stick insect embryos.

4. Discussion

The present study aimed at exploring the possibility that

vitellin cleavage products in stick insect embryos may

escape from the yolk granules and, in doing so, be

eventually targeted to the ubiquitin-dependent pathway for

completing their proteolytic degradation. Data based on

the use of several mono and polyclonal anti-vitellin

antibodies demonstrate that: (1) two major vitellin poly-

peptides, A3 and A2, are processed proteolytically to

generate cleavage products of lower molecular weight and

that (2) the proteolytic processings these polypeptides

undergo are temporally related to expression or acquisition

of an anti-ubiquitin reactivity by the yolk granules. Both

these findings suggest the possibility that processing by

limited proteolysis of vitellin A polypeptides and ubiquiti-

nation in stick insect embryos may be causally related

events. Indeed an earlier finding by Levenbook et al. (1986)

has clearly shown that ubiquitination in Calliphora vicina is

causally related to calliphorin breakdown during the insect

life cycle. The simultaneous occurrence of anti-vitellin A

and anti-ubiquitin reactivities in late vitellophages of the

stick insect embryo is highly suggestive of the possibility

that ubiquitinated proteins may gain access to the cytosolic

space of the vitellophage to be further degraded. The

labeling patterns provided by confocal analysis of these

antibodies coincide spatially with the distribution of

particulate material observed by scanning electron

microscopy on fractured yolk granule membranes (Figs.

5(E) and (F)). The origin of this material can be reasonably

traced back to vitellin cleavage products clotted by

glutaraldehyde fixation during tissue processing. Even

though artefactual, the presence of particulate material at

this cell site suggests the possibility that proteins may be

translocated from the yolk granules to the cytosolic space of

the vitellophages.

As to the nature of the ubiquitin reacting polypeptides,

several possibilities could be considered. They could be

ubiquitin-like proteins retaining some kind of homology

with ubiquitin, but be functionally unrelated to the

ubiquitin-proteasomal pathway. These proteins are referred

to as ubiquitin domain proteins. They are essentially

unrelated to each other and incapable of conjugating to

other proteins (Weisman, 2000; Muller et al., 2001). This be

the case, the presence of ubiquitin domains in vitellin

polypeptides would be hard to explain, since they could not

Fig. 7. (A) Vg polypeptides, ubiquitin-like modifiers and lysosomal pro-proteases are all stored in the same yolk granules as a result of the endocytic activity of

the oocyte during ovarian development. (B) With the onset of embryonic development yolk granules start to be acidified by intake of Hþ and ubiquitin-like

modifiers are partitioned in small granules. Upon acidification of the yolk granules, proteases are activated and, consequently, vitellin polypeptides are

processed by limited proteolysis. (C) Vitellin (Vt) cleavage products are translocated across the yolk granule membrane and ubiquinated in the cytosolic space

of the vitellophage to be degraded exhaustively by the proteasome.

A. Cecchettini et al. / Micron 34 (2003) 39–48 45

be expected to accomplish any cytosolic degradation in

stick insect embryos. Alternatively, ubiquitin reacting

polypeptides in stick insect embryos could be polypeptides

containing several lysine residues linked covalently to

ubiquitin by a terminal glycine, as they are processed during

late vitellogenesis or early embryogenesis. However, the

present study has not recorded any instance, neither during

vitellogenesis nor during embryogenesis, of any processing

event leading to an increase in relative molecular weight as

due to vitellin polypeptide ubiquitination. Since in stick

insect embryos, vitellin polypeptide A3 comes to exhibit an

anti-ubiquitin reactivity upon processing to a lower

molecular weight product, it may plausibly act as a

ubiquitin-like modifier that is transitorily prevented from

conjugating to other proteins by fusion with a carboxy-

terminal extension peptide. Hitherto, a number of such

ubiquitin-like proteins have been identified and proved to be

co-translationally processed to become exposed to ubiqui-

tin. One of these proteins was found to encode a 15 kDa

polypeptide with a significant similarity to a tandem di-

ubiquitin repeats (Haas et al., 1987). Similarly, a clone

containing nine repeats of the ubiquitin coding sequence

was isolated from an intersegmental muscle cDNA library

of the hawk-mothManduca sexta and shown to play a major

role in programmed cell death (Schwartz et al., 1990). For

the ubiquitin domains of these protein modifiers to be

exposed and to become available for interaction with other

proteins it is essential that the terminal amino acids or

peptides are removed by endoproteolytic processing

(Jentsch and Pyrowalakis, 2000). Based on these findings,

it is likely that vitellin polypeptides in stick insect embryos

may be cleaved by lysosomal proteases to generate a

number of lower molecular weight cleavage products as

long as they are inside the yolk granules (Indrasith et al.,

1987; Masetti and Giorgi, 1989) and that, following their

translocation in the cytosolic space of the vitellophages,

they may become polyubiquitinated to complete their

proteolytic degradation (Lee and Goldberg, 1998). Follow-

ing ubiquination, vitellin cleavage products may eventually

be targeted to the cytosolic proteasome 26S (Ciechanover,

2001) and be further degraded into oligopeptides or even

amino acids in a highly processive manner (Kisselev et al.,

1998). The simultaneous occurrence of lysosomal and

ubiquitin-proteasome degradation systems in insect

embryos raise the question how vitellin cleavage products

may eventually come to cross membranes enclosing the

yolk granules. The present study along with earlier evidence

(Fausto et al., 2001) has clearly shown that both yolk

granules and ubiquitin carrying granules are differentially

acidified during embryonic development. It is thus feasible

that the low pH encountered by vitellin cleavage products in

the yolk compartment may trigger translocation across the

enclosing membrane and eventually allow them to reach the

cytosolic space of the vitellophage. This interpretation is

consonant with the observation that protein unfolding, as

caused by a low luminal pH, is a prerequisite for such

proteins as the diphteria toxins to penetrate into the cytosol

(Sandvig and Olsnes, 1981; Blewitt et al., 1985; Falnes and

Sandvig, 2000). It is also compatible with a number of

recent findings showing that a multitude of resident as well

as membrane-bound proteins may be retained in the

endoplasmic reticulum if not properly folded during the

secretory process (Hiller et al., 1996; Qu et al., 1996). These

misfolded proteins are eventually destined for degradation

via the ubiquitin-proteasome system by membrane retro-

translocation, rather than by other luminal endopeptidases

(Kopito, 1998). It thus appears that a number of cell

systems, including insect embryos, may attain proteolytic

degradation of stored, membrane enclosed, proteins by

luminal acidification and retrograde transport through the

enclosing membrane. In doing so, they allow proteins to be

ubiquitinated in the cytosolic space and eventually be

degraded by the proteasome.

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