Abstract
p32 [also known as HABP1 (hyaluronan-binding protein 1), gC1qR (receptor for globular head domains complement 1q) or C1qbp (complement 1q-binding protein)] has been shown previously to have both mitochondrial and non-mitochondrial localization and functions. In the present study, we show for the first time that endogenous p32 protein is a mitochondrial protein in HeLa cells under control and stress conditions. In defining the impact of altering p32 levels in these cells, we demonstrate that the overexpression of p32 increased mitochondrial fibrils. Conversely, siRNA-mediated p32 knockdown enhanced mitochondrial fragmentation accompanied by a loss of detectable levels of the mitochondrial fusion mediator proteins Mfn (mitofusin) 1 and Mfn2. More detailed ultrastructure analysis by transmission electron microscopy revealed aberrant mitochondrial structures with less and/or fragmented cristae and reduced mitochondrial matrix density as well as more punctate ER (endoplasmic reticulum) with noticeable dissociation of their ribosomes. The analysis of mitochondrial bioenergetics showed significantly reduced capacities in basal respiration and oxidative ATP turnover following p32 depletion. Furthermore, siRNA-mediated p32 knockdown resulted in differential stress-dependent effects on cell death, with enhanced cell death observed in the presence of hyperosmotic stress or cisplatin treatment, but decreased cell death in the presence of arsenite. Taken together, our studies highlight the critical contributions of the p32 protein to the morphology of mitochondria and ER under normal cellular conditions, as well as important roles of the p32 protein in cellular metabolism and various stress responses.
Highlights
P32 [ known as HABP1, receptor for globular head domains complement 1q (gC1qR) or C1qbp], a conserved eukaryotic protein [1], was originally identified as a pre-mRNA splicing factor SF2-associated protein [2] as well as a globular protein capable of interacting with the complement component C1q [3]
The cellular architecture and the dynamic reorganization of the network are tightly controlled by a number of key proteins such as dynamin-related protein 1 (Drp-1), optical atrophy 1 (Opa1) and the Mfn 1 and Mfn2 proteins, which are found participating in mitochondrial fusion/fission machinery as well as affecting the metabolism and regulation of cell death [15,16,17]
This is the first demonstration of the requirement for the p32 protein in the maintenance of the morphology of both the mitochondrial and endoplasmic reticulum (ER) networks
Summary
P32 [ known as HABP1 (hyaluronan-binding protein 1), gC1qR (receptor for globular head domains complement 1q) or C1qbp (complement 1q-binding protein)], a conserved eukaryotic protein [1], was originally identified as a pre-mRNA splicing factor SF2-associated protein [2] as well as a globular protein capable of interacting with the complement component C1q [3]. To examine the functional roles played by p32, we evaluated mitochondrial morphology and metabolic activity after modulating p32 expression levels in HeLa cells This revealed striking changes in mitochondrial morphology, mitochondrial ATP turnover rate and basal respiration, and indicated for the Abbreviations used: CCCP, carbonyl cyanide m-chlorophenylhydrazone; DMEM, Dulbecco’s modified Eagle’s medium; Drp-1, dynamin-related protein 1; ECAR, extracellular acidification rate; ER, endoplasmic reticulum; FCCP, carbonyl cyanide p-trifluoromethoxyphenylhydrazone; gC1qR, receptor for globular head domains complement 1q; JNK, c-Jun N-terminal kinase; LDH, lactate dehydrogenase; Mfn, mitofusin; OCR, oxygen consumption rate; Opa, optical atrophy 1; p32 Nm-terminus, N-terminus of mature p32 protein; PARP, poly(ADP-ribose) polymerase; PJNK, phosphorylated c-Jun N-terminal kinase; RIPA, radioimmunoprecipitation assay; TBST, 10 mM Tris/HCl, pH 7.5, 150 mM NaCl and 0.1 % Tween 20; TEM, transmission electron microscopy; XTT, sodium 3 -[1-(phenylaminocarbonyl)-3,4-tetrazolium]-bis (4-methoxy-6-nitro) benzene sulfonic acid hydrate
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