Affiliation: National Heart Lung and Blood Institutes, NIH
Keywords: a. spermatogenesis; d. centrosome
Centrosomes are the major microtubule organizing centers (MTOC) that are composed of centrioles and the pericentriolar material (PCM). Centrosomes are essential part of diverse cellular processes that require precise regulation of their protein levels. One protein whose levels must be regulated is Pericentrin – PCNT in humans and PLP in Drosophila. Increased PCNT expression and its protein accumulation are linked to many clinical conditions especially in patients diagnosed with cancer, mental disorders, and ciliopathies reflecting the requirement for regulating PCNT level. However, the mechanisms by which the PCNT is regulated remain less explored. In this study, we took advantage of Drosophila spermatogenesis to characterize the mechanistic of PCNT/PLP degradation. Our previous study by Galletta, 2020 demonstrated that PLP levels are sharply downregulated during early spermatogenesis and this regulation is essential to spatially position PLP in the meiotic centrioles. We therefore first performed a structure-function analysis to identify the functional domain required for regulating PLP level and localization. We found that the N-terminal region of PLP is essential to regulate its protein level and depletion of these regions led to PLP stabilization, which in turn mislocalized PLP on the centrioles. As a consequent, the PCM was also mispositioned leading to defects in spermatids, which ultimately compromised the sperm function. Our biochemical and proteomic analysis revealed that the N terminal region of PLP harbors degradation signals and engages interactions with multiple proteosomal components that hinted a precise degradation process to act via N terminal region. To identify the mechanisms, we performed a candidate RNAi screen composed of proteasome components. We found Rad6, UbcD1 and its related UBR box family E3 ligases, Poe (UBR4) and Hyd (UBR5) to promote PLP degradation in spermatocytes and therefore regulate its spatial localization on the centrioles. The identified candidates are known regulators of the N-end rule degradation pathway, in which their substrates are degraded based on the characteristics of the N-termini amino acids that require single or sequential enzymatic mortification to form N-degron. By characterizing the N terminal amino acids, we found that PLP is a potential substrate of the N-end rule pathway. Collectively, our study identified N-end rule pathway to regulate PLP levels for proper centrosome assembly and may also be relevant for PCNT homeostasis in human as well.