Targeted Mutation of the MLN64 START Domain Causes Only Modest Alterations in Cellular Sterol Metabolism

Abstract

The StAR-related lipid transfer (START) domain, first identified in the steroidogenic acute regulatory protein (StAR), is involved in the intracellular trafficking of lipids. Sixteen mammalian START domain-containing proteins have been identified to date. StAR, a protein targeted to mitochondria, stimulates the movement of cholesterol from the outer to the inner mitochondrial membranes, where it is metabolized into pregnenolone in steroidogenic cells. MLN64, the START domain protein most closely related to StAR, is localized to late endosomes along with other proteins involved in sterol trafficking, including NPC1 and NPC2, where it has been postulated to participate in sterol distribution to intracellular membranes. To investigate the role of MLN64 in sterol metabolism, we created mice with a targeted mutation in the Mln64 START domain, expecting to find a phenotype similar to that in humans and mice lacking NPC1 or NPC2 (progressive neurodegenerative symptoms, free cholesterol accumulation in lysosomes). Unexpectedly, mice homozygous for the Mln64 mutant allele were viable, neurologically intact, and fertile. No significant alterations in plasma lipid levels, liver lipid content and distribution, and expression of genes involved in sterol metabolism were observed, except for an increase in sterol ester storage in mutant mice fed a high fat diet. Embryonic fibroblast cells transfected with the cholesterol side-chain cleavage system and primary cultures of granulosa cells from Mln64 mutant mice showed defects in sterol trafficking as reflected in reduced conversion of endogenous cholesterol to steroid hormones. These observations suggest that the Mln64 START domain is largely dispensable for sterol metabolism in mice.

Overexpression of the cholesterol-binding protein MLN64 induces liver damage in the mouse

“…Removal of the MLN64 N-terminal region increased steroidogenesis in COS-1 transfected cells [16] and enhanced the generation of steroid hormone by placental mitochondria in in vitro assays [8] . However, mice with targeted mutation of the MLN64 START domain did not exhibit a sterol-metabolism-related phenotype [15] , questioning the physiological importance of the MLN64 START domain in cholesterol trafficking and metabolism.…”

“…However, the overexpression of full-length MLN64 also induced an increase in sterol accumulation in COS cells, arguing against a role for MLN64 in cholesterol efflux from this compartment [14] . Moreover, mice with targeted mutation of the MLN64 START domain were neurologically intact and fertile, and exhibited only modest alterations in cellular sterol metabolism [15] , leaving unanswered the question of MLN64 function in vivo.…”

Niemann-Pick Type C Disease and Intracellular Cholesterol Trafficking

“…The role of MLN64 in sterol trafficking is not clear because mice with targeted mutation of MLN64 are healthy and display only minimal disturbances in sterol dynamics (47). A novel CHO cell mutant without the NPC1 mutation but with defects in late endosomal cholesterol trafficking has been isolated (48).…”

The mammalian START domain protein family in lipid transport in health and disease

“…The MENTAL domain of STARD3 is capable of binding cholesterol and is required for its dimerization with another endosomal membrane protein composed only of a MENTAL domain termed MENTHO (MLN64 N-terminal homolog; ). In one model for trafficking of late endosome/lysosome cholesterol, STARD3/MLN64 acts as the cytosolic acceptor of NPC1-derived cholesterol (reviewed in Strauss et al (2003) (Kishida et al 2004). Homozygous STARD3/ MLN64 mutant mice that express a STARD3/MLN64 protein containing the N-terminal MENTAL domain but lacking the START domain do not accumulate cholesterol in late endosomes/lysosomes and synthesize steroid hormones at wild-type levels (Kishida et al 2004).…”

“…In one model for trafficking of late endosome/lysosome cholesterol, STARD3/MLN64 acts as the cytosolic acceptor of NPC1-derived cholesterol (reviewed in Strauss et al (2003) (Kishida et al 2004). Homozygous STARD3/ MLN64 mutant mice that express a STARD3/MLN64 protein containing the N-terminal MENTAL domain but lacking the START domain do not accumulate cholesterol in late endosomes/lysosomes and synthesize steroid hormones at wild-type levels (Kishida et al 2004). Stard4, Stard5, Npc1, and Npc2 mRNA levels were not changed due to loss of the START domain from STARD3/MLN64, suggesting that the lack of a phenotype was not due to compensatory increases of these cholesterol transporters.…”