Exclusion of germ plasm proteins from somatic lineages by cullin-dependent degradation

Abstract

In many animals, establishment of the germ line depends on segregation of a specialized cytoplasm, or 'germ plasm', to a small number of germline precursor cells during early embryogenesis. Germ plasm asymmetry involves targeting of RNAs and proteins to a specific region of the oocyte and/or embryo. Here we demonstrate that germ plasm asymmetry also depends on degradation of germline proteins in non-germline (somatic) cells. We show that five CCCH finger proteins, components of the Caenorhabditis elegans germ plasm, are targeted for degradation by the novel CCCH-finger-binding protein ZIF-1. ZIF-1 is a SOCS-box protein that interacts with the E3 ubiquitin ligase subunit elongin C. Elongin C, the cullin CUL-2, the ring finger protein RBX-1 and the E2 ubiquitin conjugation enzyme UBC5 (also known as LET-70) are all required in vivo for CCCH finger protein degradation. Degradation is activated in somatic cells by the redundant CCCH finger proteins MEX-5 and MEX-6, which are counteracted in the germ line by the PAR-1 kinase. We propose that segregation of the germ plasm involves both stabilization of germline proteins in the germ line and cullin-dependent degradation in the soma.

Figure 1

ZIF-1 interacts with CCCH fingers targeted for degradation. a, Early embryonic lineage. P₁ to P₄ are the germline blastomeres. AB, EMS, C and D are the somatic blastomeres. PAR-1 (green), PIE-1 (red) and MEX-5 (blue) segregations are indicated; line thickness indicates relative levels. b, Yeast transformed with ZIF-1 and finger domains from indicated proteins. ZF1m and ZF2m are SSCH instead of CCCH. Growth indicates interaction. WT, wild type. c, Labelled PIE-1 proteins (full-length, lanes 1–3; ZF1 deleted, lanes 4–6) synthesized in rabbit reticulolysates and incubated with E. coli-synthesized GST (lanes 2 and 5) or GST–ZIF-1 (lanes 3 and 6) on Sepharose beads. Bound proteins were eluted and run on SDS–PAGE. 100% of input (lanes 1 and 4) was loaded on GST column; 50% of bound was loaded on gel.

Figure 2

Depletion of ZIF-1 blocks degradation of CCCH finger proteins in somatic cells, but does not affect other soma–germline asymmetries. a–f, 28-cell wild-type and zif-1(RNAi) embryos expressing PIE-1::GFP (a), POS-1 (b, immunofluorescence5), GFP::MEX-5 (c), GFP::PIE-1 with ZF1 deleted (d), GFP::PAR-2 (e) and pie-1 RNA (f, in situ hybridization27). Measurement of GFP fluorescence (IP lab software) in ABa (soma) confirmed that PIE-1::GFP levels are higher in zif-1(RNAi) compared with wild type (t-test; P = 0.009), but do not change significantly in P₂ (germ line) (t-test; P = 0.25, N = 10 four-cell embryos for each genotype). g, One-cell wild-type and zif-1(RNAi) embryos expressing a PIE-1::GFP fusion. In later stages, the zif-1(RNAi) embryo failed to degrade PIE-1::GFP in somatic blastomeres (see ftp://ftp.wormbase.org/pub/wormbase/datasets/derenzo_2003), confirming the efficient inactivation of zif-1.

Figure 3

ZIF-1 is a SOCS-box protein. Alignment of the ZIF-1 SOCS box with SOCS boxes from other elongin C-interacting proteins. Asterisk indicates cysteine mutated in Fig. 4a. ce, C. elegans; hs, Homo sapiens; mm, Mus musculus.

Figure 4

ZIF-1 may function as a substrate-recruitment factor for an elongin C/CUL-2 E3 ubiquitin ligase. a, Yeasts expressing ZIF-1, or ZIF-1 with a SOCS mutation, and elongin C or PIE-1 as indicated. Growth indicates interaction. b, Diagram after ref. depicting ECS ligase with its associated E2, substrate-recruitment subunits (ZIF-1/VHL) and substrates (PIE-1/HIF1α). c, Four-cell embryos expressing GFP::PIE-1^ZF1 or GFP::PGL-1 and partially depleted by RNAi for the indicated genes. Visualization of PGL-1 and GFP::PIE-1^ZF1 in the same cul-2(RNAi) embryos confirmed that partial cul-2 depletion blocks degradation without affecting overall polarity (data not shown). d, Live embryos (outlined) expressing GFP::ZIF-1, driven by the pie-1 promoter (Methods), in wild-type mothers or mothers exposed to cul-2 or cul-3 double-stranded RNA. Punctate fluorescence below the embryos is gut autofluorescence from the mother.

Figure 5

PAR-1 inhibits, and MEX-5 and MEX-6 activate, ZIF-1-dependent degradation. a, 12/15-cell embryos expressing GFP::PIE-1^ZF1 with genes inactivated by RNAi. GFP::PIE-1^ZF1 is degraded in somatic blastomeres in wild type, and in all cells in par-1(RNAi) embryos. Ubiquitous degradation in par-1(RNAi) requires ZIF-1, MEX-5, MEX-6 and CUL-2. Similar results were obtained with PIE-1::GFP (Supplementary Information). b, Schematic summarizing the epistatic relationships deduced from a and ref. .