PERK recruits E-Syt1 at ER-mitochondria contacts for mitochondrial lipid transport and respiration.

The integrity of ER-mitochondria appositions ensures transfer of ions and phospholipids (PLs) between these organelles and exerts crucial effects on mitochondrial bioenergetics. Malfunctions within the ER-mitochondria contacts altering lipid trafficking homeostasis manifest in diverse pathologies, but the molecular effectors governing this process remain ill-defined. Here, we report that PERK promotes lipid trafficking at the ER-mitochondria contact sites (EMCS) through a non-conventional, unfolded protein response-independent, mechanism. PERK operates as an adaptor for the recruitment of the ER-plasma membrane tether and lipid transfer protein (LTP) Extended-Synaptotagmin 1 (E-Syt1), within the EMCS. In resting cells, the heterotypic E-Syt1-PERK interaction endorses transfer of PLs between the ER and mitochondria. Weakening the E-Syt1-PERK interaction or removing the lipid transfer SMP-domain of E-Syt1, compromises mitochondrial respiration. Our findings unravel E-Syt1 as a PERK interacting LTP and molecular component of the lipid trafficking machinery of the EMCS, which critically maintains mitochondrial homeostasis and fitness.

ORP5/8 and MIB/MICOS link ER-mitochondria and intra-mitochondrial contacts for non-vesicular transport of phosphatidylserine.

Mitochondria are dynamic organelles essential for cell survival whose structural and functional integrity rely on selective and regulated transport of lipids from/to the endoplasmic reticulum (ER) and across the mitochondrial intermembrane space. As they are not connected by vesicular transport, the exchange of lipids between ER and mitochondria occurs at membrane contact sites. However, the mechanisms and proteins involved in these processes are only beginning to emerge. Here, we show that the main physiological localization of the lipid transfer proteins ORP5 and ORP8 is at mitochondria-associated ER membrane (MAM) subdomains, physically linked to the mitochondrial intermembrane space bridging (MIB)/mitochondrial contact sites and cristae junction organizing system (MICOS) complexes that bridge the two mitochondrial membranes. We also show that ORP5/ORP8 mediate non-vesicular transport of phosphatidylserine (PS) lipids from the ER to mitochondria by cooperating with the MIB/MICOS complexes. Overall our study reveals a physical and functional link between ER-mitochondria contacts involved in lipid transfer and intra-mitochondrial membrane contacts maintained by the MIB/MICOS complexes.

Chemical targeting of NEET proteins reveals their function in mitochondrial morphodynamics.

Several human pathologies including neurological, cardiac, infectious, cancerous, and metabolic diseases have been associated with altered mitochondria morphodynamics. Here, we identify a small organic molecule, which we named Mito-C. Mito-C is targeted to mitochondria and rapidly provokes mitochondrial network fragmentation. Biochemical analyses reveal that Mito-C is a member of a new class of heterocyclic compounds that target the NEET protein family, previously reported to regulate mitochondrial iron and ROS homeostasis. One of the NEET proteins, NAF-1, is identified as an important regulator of mitochondria morphodynamics that facilitates recruitment of DRP1 to the ER-mitochondria interface. Consistent with the observation that certain viruses modulate mitochondrial morphogenesis as a necessary part of their replication cycle, Mito-C counteracts dengue virus-induced mitochondrial network hyperfusion and represses viral replication. The newly identified chemical class including Mito-C is of therapeutic relevance for pathologies where altered mitochondria dynamics is part of disease etiology and NEET proteins are highlighted as important therapeutic targets in anti-viral research.

PMEL Amyloid Fibril Formation: The Bright Steps of Pigmentation.

In pigment cells, melanin synthesis takes place in specialized organelles, called melanosomes. The biogenesis and maturation of melanosomes is initiated by an unpigmented step that takes place prior to the initiation of melanin synthesis and leads to the formation of luminal fibrils deriving from the pigment cell-specific pre-melanosomal protein (PMEL). In the lumen of melanosomes, PMEL fibrils optimize sequestration and condensation of the pigment melanin. Interestingly, PMEL fibrils have been described to adopt a typical amyloid-like structure. In contrast to pathological amyloids often associated with neurodegenerative diseases, PMEL fibrils represent an emergent category of physiological amyloids due to their beneficial cellular functions. The formation of PMEL fibrils within melanosomes is tightly regulated by diverse mechanisms, such as PMEL traffic, cleavage and sorting. These mechanisms revealed increasing analogies between the formation of physiological PMEL fibrils and pathological amyloid fibrils. In this review we summarize the known mechanisms of PMEL fibrillation and discuss how the recent understanding of physiological PMEL amyloid formation may help to shed light on processes involved in pathological amyloid formation.

Restricted Location of PSEN2/γ-Secretase Determines Substrate Specificity and Generates an Intracellular Aß Pool.

γ-Secretases are a family of intramembrane-cleaving proteases involved in various signaling pathways and diseases, including Alzheimer's disease (AD). Cells co-express differing γ-secretase complexes, including two homologous presenilins (PSENs). We examined the significance of this heterogeneity and identified a unique motif in PSEN2 that directs this γ-secretase to late endosomes/lysosomes via a phosphorylation-dependent interaction with the AP-1 adaptor complex. Accordingly, PSEN2 selectively cleaves late endosomal/lysosomal localized substrates and generates the prominent pool of intracellular Aß that contains longer Aß; familial AD (FAD)-associated mutations in PSEN2 increased the levels of longer Aß further. Moreover, a subset of FAD mutants in PSEN1, normally more broadly distributed in the cell, phenocopies PSEN2 and shifts its localization to late endosomes/lysosomes. Thus, localization of γ-secretases determines substrate specificity, while FAD-causing mutations strongly enhance accumulation of aggregation-prone Aß42 in intracellular acidic compartments. The findings reveal potentially important roles for specific intracellular, localized reactions contributing to AD pathogenesis.

Parkinson's Disease: A Traffic Jam?

Recent large-scale proteomic analyses of two protein kinases that are linked to Parkinson's disease have identified a remarkable convergence between their respective impacts on the phosphoproteome: activation of both LRRK2 and PINK1 leads to phosphorylation of several members of the Rab family of small GTPases, which regulate membrane trafficking.

BACE2 processes PMEL to form the melanosome amyloid matrix in pigment cells.

Amyloids are often associated with pathologic processes such as in Alzheimer's disease (AD), but can also underlie physiological processes such as pigmentation. Formation of pathological and functional amyloidogenic substrates can require precursor processing by proteases, as exemplified by the generation of Aß peptide from amyloid precursor protein (APP) by beta-site APP cleaving enzyme (BACE)1 and γ-secretase. Proteolytic processing of the pigment cell-specific Melanocyte Protein (PMEL) is also required to form functional amyloid fibrils during melanogenesis, but the enzymes involved are incompletely characterized. Here we show that the BACE1 homologue BACE2 processes PMEL to generate functional amyloids. BACE2 is highly expressed in pigment cells and Bace2(-/-) but not Bace1(-/-) mice display coat color defects, implying a specific role for BACE2 during melanogenesis. By using biochemical and morphological analyses, combined with RNA silencing, pharmacologic inhibition, and BACE2 overexpression in a human melanocytic cell line, we show that BACE2 cleaves the integral membrane form of PMEL within the juxtamembrane domain, releasing the PMEL luminal domain into endosomal precursors for the formation of amyloid fibrils and downstream melanosome morphogenesis. These studies identify an amyloidogenic substrate of BACE2, reveal an important physiological role for BACE2 in pigmentation, and highlight analogies in the generation of PMEL-derived functional amyloids and APP-derived pathological amyloids.

The tetraspanin CD63 regulates ESCRT-independent and -dependent endosomal sorting during melanogenesis.

Cargo sorting to intraluminal vesicles (ILVs) of multivesicular endosomes is required for lysosome-related organelle (LRO) biogenesis. PMEL-a component of melanocyte LROs (melanosomes)-is sorted to ILVs in an ESCRT-independent manner, where it is proteolytically processed and assembled into functional amyloid fibrils during melanosome maturation. Here we show that the tetraspanin CD63 directly participates in ESCRT-independent sorting of the PMEL luminal domain, but not of traditional ESCRT-dependent cargoes, to ILVs. Inactivating CD63 in cell culture or in mice impairs amyloidogenesis and downstream melanosome morphogenesis. Whereas CD63 is required for normal PMEL luminal domain sorting, the disposal of the remaining PMEL transmembrane fragment requires functional ESCRTs but not CD63. In the absence of CD63, the PMEL luminal domain follows this fragment and is targeted for ESCRT-dependent degradation. Our data thus reveal a tight interplay regulated by CD63 between two distinct endosomal ILV sorting processes for a single cargo during LRO biogenesis.