A C-Terminal Transmembrane Anchor Targets the Nuage-Localized Spermatogenic Protein Gasz to the Mitochondrial Surface

Abstract

Mitochondria, normally tubular and distributed throughout the cell, are instead found in spermatocytes in perinuclear clusters in close association with nuage, an amorphous organelle composed of RNA and RNA-processing proteins that generate piRNAs. piRNAs are a form of RNAi required for transposon suppression and ultimately fertility. MitoPLD, another protein required for piRNA production, is anchored to the mitochondrial surface, suggesting that the nuage, also known as intermitochondrial cement, needs to be juxtaposed there to bring MitoPLD into proximity with the remainder of the piRNA-generating machinery. However, the mechanism underlying the juxtaposition is unknown. Gasz, a multidomain protein of known function found in the nuage in vertebrates, is required for piRNA production and interacts with other nuage proteins involved in this pathway. Unexpectedly, we observed that Gasz, in nonspermatogenic mammalian cells lines, localizes to mitochondria and does so through a previously unrecognized conserved C-terminal mitochondrial targeting sequence. Moreover, in this setting, Gasz is able to recruit some of the normally nuage-localized proteins to the mitochondrial surface. Taken together, these findings suggest that Gasz is a nuage-localized protein in spermatocytes that facilitates anchoring of the nuage to the mitochondrial surface where piRNA generation takes place as a collaboration between nuage and mitochondrial-surface proteins.

GASZ and mitofusin‐mediated mitochondrial functions are crucial for spermatogenesis

“…By contrast, mutated MLS (Phe/F to Pro/P mutation in MLS, GFP‐F/P) or MLS with the deletion of its last four amino acids (ΔLQRK) failed to bring GFP to mitochondria (Fig C). These results, in large part, match those found in recent findings by Altshuller et al . In common, we identified a functional MLS at the C‐terminus of GASZ protein, and its mutation leads to the mislocalization of GASZ from mitochondria to the cytoplasm in somatic cells.…”

“…GASZ may act as a central scaffold protein to recruit these germ cell‐specific factors into close proximity at mitochondria at IMC. To support this hypothesis, multiple nuage‐associated proteins (including MIWI, DAZL, MVH, and TDRD1) have been previously identified as interacting partners for GASZ . Unlike a reported GASZ‐knockout study , GASZ and MILI proteins were still found in the GASZ Δ MLS/ Δ MLS gonocytes with the MLS deletion, and both of them displayed a diffused pattern in the cytoplasm instead of a perinuclear granular localization at mitochondria in wild‐type controls.…”

Mitochondria Associated Germinal Structures in Spermatogenesis: piRNA Pathway Regulation and Beyond

“…Notably, whether GASZ localizes mitochondria outer membrane depends on its C-terminal mitochondrial targeting sequences and is mainly co-expressed with IMC component MILI both in the gonocytes and spermatocytes, and partially co-localizes with MVH and TDRD1, indicating its enrichment in IMC. In contrast, in round spermatid, GASZ is still expressed as a granular signal in the cytoplasm partially co-staining with mitochondria, but not localized in the CB [31,46,49].…”

GASZ self-interaction clusters mitochondria into the intermitochondrial cement for proper germ cell development

“…It remains unclear how IMC forms in embryonic prospermatogonia and postnatal spermatogonia to support spermatogenesis. GASZ is highly expressed at embryonic day 13.5 ( 29 ), and is the only known germ cell-specific mitochondrial protein that is expressed when IMC starts to appear and affects IMC integrity when deleted from embryonic germ cells ( 23 , 27 ). In this study, we found that GASZ formed dimers and were able to cluster mitochondria in somatic cells when no other germ cell-specific proteins were present.…”

“…To date, only a few germ cell-specific proteins are known to be localized directly at mitochondria. Of these, GASZ (also called ASZ1) is found at the mitochondrial outer membrane via a C-terminal mitochondrial localization signal (MLS) ( 23 , 27 ). GASZ is a germ cell-specific protein with four Ankyrin repeats (ANK), a sterile alpha motif (SAM), and a putative basic leucine zipper domain (bZIP) ( 28 ).…”