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First published online 23 January 2003
doi: 10.1242/jcs.00332

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Deletion of proteasomal subunit S5a/Rpn10/p54 causes lethality, multiple mitotic defects and overexpression of proteasomal genes in Drosophila melanogaster

Biological Research Center of the Hungarian Academy of Sciences, H-6701 Szeged, P.O. Box 521, Hungary

View larger version (14K): [in a new window]   Fig. 1. Molecular map of the pros54 genomic region. (A) Position of the pros54, Vha M9.7-2 and CG7181 genes according to GadFly. Arrows show the location and the direction of these genes. The triangle labels the site of P-lacW0554/18 insertion. The E and PIR PCR primers used to screen for genomic deletions are indicated in the upper part of the figure. S, E and P are SacI, EcoRI and PstI restriction sites, respectively. Numbers in parentheses give the nucleotide-scale positions according to GadFly. (B) Extension of Df(3L)pros54P(w+). (C) Restriction fragments used in rescue experiments (see text).

View larger version (71K): [in a new window]   Fig. 2. Pupal lethal phenotype of p54 mutant. (A) 60-hour-old (APF) puparia of p54 (top and middle) and Oregon R wild-type (bottom). The mutant puparia are characteristically bent and smaller than the wild-type. (B) 60-hour-old (APF) puparia of p54 (top: side view, bottom: dorsal view). The pupae inside are separated from the puparial cuticle (see also A). In the bottom animal the pupal cuticle was laid down in the head and tail regions but remained open in the middle of the body and the internal tissues are exposed (arrow). (C-F) Pupae removed from the puparial case for comparison. (C) In the 16-hour-old (APF) wild-type pupa the main body parts of the adult (head, thorax, abdomen) are formed and the wings and legs everted. (D) In the 60-hour-old mutant pupa the head and thorax are significantly smaller and the appendages shorter than those of the 16-hour-old wildtype. Adult cuticle secretion and eye pigment deposition were never observed. (E) 60-hour-old wild-type pupa. The hypoderm already separated from the pupal cuticle (arrowhead) in preparation for the adult cuticle secretion. Pigment deposition is visible in the eyes. (F) A 60-hour-old (APF) mutant pupa with minimal signs of development: the pupal cuticle can be found only in the regions of the head and the external genitalia (arrowheads). On the other parts of the body the internal tissues are exposed. D and F represent the two extremes of the p54 mutant phenotype (see text for details). A,C,E are anterior to the left; B,D,F are anterior to the right. (G) Larval brain from mutant and wild-type wandering larvae.

View larger version (135K): [in a new window]   Fig. 3. Mitotic figures from wild-type and p54 mutant larval brains. Aceto-orcein-stained metaphase figures from wild-type (A) and p54 (B-F) third instar larval brains. p54 mitotic cells frequently show highly condensed chromosomes (B), aneuploid (B,E) or polyploid (D) chromosome sets and circular mitotic figures (C). Characteristically, some cells appear to prematurely separate their sister chromatids (F).

View larger version (64K): [in a new window]   Fig. 4. Complete lack of subunit S5a/Rpn10/p54 in p54 mutant pupae. Total protein extracts of wild-type (lane 1) and p54 (lane 2) pupae were fractionated on 8% SDS-PAGE and immunoblotted with a mixture of four different monoclonal antibodies specific for subunits p54, p48A, p42C and p39.

View larger version (15K): [in a new window]   Fig. 5. Developmental profile of the 26S proteasome in the Oregon R wild-type strain. Total protein extract from 0- to 24-hour-old embryos (lane 1), 3rd instar larvae (lane 2), 0-hour-old (lane 3), 2-hour-old (lane 4), 4-hour-old (lane 5), 6-hour-old (lane 6), 8-hour-old (lane 7), 10-hour-old (lane 8) prepupae as well as 12-hour-old (lane 9), 18-hour-old (lane 10) and 24-hour-old (lane 11) pupae were fractionated on 8% SDS-PAGE and immunoblotted with a mixture of two different monoclonal antibodies specific for subunits p54 and p48A. The age of the specimens is given in hours after white puparium formation.

View larger version (24K): [in a new window]   Fig. 6. Multiubiquitinated protein profile in wild-type and p54 pupae. Total protein extracts prepared from a single wild-type (lane 1) or a single p54 (lane 2) pupa (20 hours APF) were fractionated on 9% SDS-PAGE and immunoblotted with a polyclonal anti-ubiquitin antibody.

View larger version (67K): [in a new window]   Fig. 7. 26S proteasomes in wild-type and p54 pupae analysed by native polyacrylamide gel electrophoresis. Total protein extracts prepared from wild-type (lanes 1, 3 and 5) or p54 (lanes 2, 4 and 6) pupae (20 hours APF) were fractionated on native polyacrylamide gel and immunoblotted with a monoclonal antibody specific for subunit p42C present in the base subcomplex (lanes 1 and 2), with a monoclonal antibody specific for a subunit p39 present in the lid subcomplex (lanes 3 and 4) or with a polyclonal antibody specific for the 20S proteasome, the catalytic core (lanes 5 and 6).

View larger version (49K): [in a new window]   Fig. 8. Accumulation of proteasomal proteins in p54 animals. Total protein extracts prepared from a single wild-type (lane 1) or a single p54 (lane 2) pupa (20 hours APF) were fractionated on 10% SDS-PAGE and immunoblotted with a polyclonal antibody specific for the catalytic core. The same extracts fractionated on a 8% SDS-PAGE were immunoblotted with the following polyclonal antibodies: anti-regulatory complex antibody (lanes 3 and 4), anti-glycogen phosphorylase antibody (lanes 5 and 6) and anti-karyopherin ß antibody (lanes 7 and 8).