AKIR-1 regulates proteasome subcellular function in Caenorhabditis elegans.

Polyubiquitinated proteins are primarily degraded by the ubiquitin-proteasome system (UPS). Proteasomes are present both in the cytoplasm and nucleus. Here, we investigated mechanisms coordinating proteasome subcellular localization and activity in a multicellular organism. We identified the nuclear protein-encoding gene akir-1 as a proteasome regulator in a genome-wide Caenorhabditis elegans RNAi screen. We demonstrate that depletion of akir-1 causes nuclear accumulation of endogenous polyubiquitinated proteins in intestinal cells, concomitant with slower in vivo proteasomal degradation in this subcellular compartment. Remarkably, akir-1 is essential for nuclear localization of proteasomes both in oocytes and intestinal cells but affects differentially the subcellular distribution of polyubiquitinated proteins. We further reveal that importin ima-3 genetically interacts with akir-1 and influences nuclear localization of a polyubiquitin-binding reporter. Our study shows that the conserved AKIR-1 is an important regulator of the subcellular function of proteasomes in a multicellular organism, suggesting a role for AKIR-1 in proteostasis maintenance.

Immunohistochemical analysis reveals variations in proteasome tissue expression in C. elegans.

The ubiquitin-proteasome system (UPS) plays a crucial part in normal cell function by mediating intracellular protein clearance. We have previously shown that UPS-mediated protein degradation varies in a cell type-specific manner in C. elegans. Here, we use formalin-fixed, paraffin-embedded C. elegans sections to enable studies on endogenous proteasome tissue expression. We show that the proteasome immunoreactivity pattern differs between cell types and within subcellular compartments in adult wild-type (N2) C. elegans. Interestingly, widespread knockdown of proteasome subunits by RNAi results in tissue-specific changes in proteasome expression instead of a uniform response. In addition, long-lived daf-2(e1370) mutants with impaired insulin/IGF-1 signaling (IIS) display similar proteasome tissue expression as aged-matched wild-type animals. Our study emphasizes the importance of alternate approaches to the commonly used whole animal lysate-based methods to detect changes in proteasome expression occurring at the sub-cellular, cell or tissue resolution level in a multicellular organism.

RAB24 facilitates clearance of autophagic compartments during basal conditions.

RAB24 belongs to a family of small GTPases and has been implicated to function in autophagy. Here we confirm the intracellular localization of RAB24 to autophagic vacuoles with immuno electron microscopy and cell fractionation, and show that prenylation and guanine nucleotide binding are necessary for the targeting of RAB24 to autophagic compartments. Further, we show that RAB24 plays a role in the maturation and/or clearance of autophagic compartments under nutrient-rich conditions, but not during short amino acid starvation. Quantitative electron microscopy shows an increase in the numbers of late autophagic compartments in cells silenced for RAB24, and mRFP-GFP-LC3 probe and autophagy flux experiments indicate that this is due to a hindrance in their clearance. Formation of autophagosomes is shown to be unaffected by RAB24-silencing with siRNA. A defect in aggregate clearance in the absence of RAB24 is also shown in cells forming polyglutamine aggregates. This study places RAB24 function in the termination of the autophagic process under nutrient-rich conditions.