While it has been appreciated for decades that lysosomes can import cysteine, its for organismal physiology is unclear. Recently, the MFSD12 transmembrane protein was shown to be necessary to import cysteine into lysosomes (and melanosomes), enabling the study of these processes using genetic tools. Here, we find that mice lacking Mfsd12 die between embryonic days 10.5-12.5, suggesting that MFSD12 is essential for organogenesis. Within lysosomes, it is well known that a significant fraction of the cysteine is converted into cystine (oxidized cysteine), which accumulates and can be released into the cytosol via the CTNS (cystinosin) transporter. However, in contrast to Mfsd12 , loss of Ctns results in live animals, indicating that it is not an essential gene. This suggests that the essential function of MFSD12 is not to enable cystine storage for use in the cytosol but rather to supply reduced cysteine to the lysosomal lumen itself. Consistent with this idea, the treatment of breeding heterozygous mice with cysteamine, a lysosome-penetrant thiol, rescued the development of Mfsd12 knockout mice and resulted in live births. Our work implicates lysosomal thiol import as an essential metabolic pathway and provides tools for deciphering its complex genetic and metabolic interactions.

Skin aging is characterized by epidermal atrophy, reduced keratinocyte proliferation, and the accumulation of senescent cells that sustain a chronic, low-grade inflammatory secretome known as the senescence-associated secretory phenotype (SASP). We previously showed that Y-box binding protein 1 (YBX1) limits keratinocyte senescence in human epidermis by acting as a translational repressor of SASP chemokines, including CXCL1 and IL8. How this brake is regulated during skin aging, however, remains undefined. Here we demonstrate that, although total YBX1 protein is reduced in keratinocytes from aged human epidermis, the fraction of phosphorylated YBX1 (pYBX1) is increased relative to young donors, resulting in an elevated pYBX1/total YBX1 ratio that correlates with chronological age. Because pYBX1 is predominantly nuclear, whereas unphosphorylated YBX1 is cytoplasmic, this shift is predicted to deplete the cytoplasmic pool available for translational repression of CXCL1 and IL8. Consistent with this model, treatment of immortalized (Ker-CT) and primary human keratinocytes (HK) with the PI3K inhibitors PI-103 and GDC-0941 suppressed YBX1 phosphorylation without altering total YBX1 abundance, thus retaining YBX1 in the cytoplasm. This allows cytoplasmic YBX1 to re-engage and repress the translation of CXCL1 and IL8 mRNAs, lower chemokine secretion, and reduce the fraction of senescent keratinocytes. Moreover, conditioned medium from PI-103- or GDC-0941-treated cells sufficiently decreased senescence in recipient cells, consistent with a paracrine, secretome-mediated effect. Together, these findings support an association between YBX1 phosphorylation and SASP chemokine output in aging human keratinocytes and nominate YBX1-directed modulation as a candidate strategy for selectively attenuating senescence-associated secretory phenotypes in intrinsic skin aging.