A Diffraction-Quality Protein Crystal Processed as an Autophagic Cargo

Abstract

Crystallization of proteins may occur in the cytosol of a living cell, but how a cell responds to intracellular protein crystallization remains unknown. We developed a variant of coral fluorescent protein that forms diffraction-quality crystals within mammalian cells. This expression system allowed the direct determination of its crystal structure at 2.9 Å, as well as observation of the crystallization process and cellular responses. The micron-sized crystal, which emerged rapidly, was a pure assembly of properly folded β-barrels and was recognized as an autophagic cargo that was transferred to lysosomes via a process involving p62 and LC3. Several lines of evidence indicated that autophagy was not required for crystal nucleation or growth. These findings demonstrate that in vivo protein crystals can provide an experimental model to study chemical catalysis. This knowledge may be beneficial for structural biology studies on normal and disease-related protein aggregation.

Ubiquitin-Dependent And Independent Signals In Selective Autophagy

“…The autophagosome fuses with late endosomes and lysosomes to generate the autolysosome that degrades and recycles the enclosed content. nucleic acids, zymogen granules (zymophagy), stress granules (granulophagy), the midbody ring, signaling (signalophagy) and biosynthetic [fatty acid synthase (FAS) degradation] protein complexes, proteasome (proteaphagy), viral components (virophagy), myelin (myelinophagy), or even synaptic vesicles and protein crystals, indicating versatility of selective autophagy and diversity of autophagy receptors [6,11,[15][16][17][18][19][20][21][22][23][24][25] (Table 1). Autophagy receptors interact with autophagosomes via LC3-interacting regions (LIR) that share the evolutionary conserved consensus sequence Trp/Phe/Tyr-x-x-Leu/Ile/Val (W/F/YxxL/I/V), and their activity is tightly controlled to ensure efficient, precise, and timely removal of substrates [6,26].…”

Giant crystals inside mitochondria of equine chondrocytes

“…Most literature data of intramitochondrial crystals in general are from the booming period of ultrastructure, the 1970s, which is probably due to the fact that crystal formation in mitochondria could not be experimentally manipulated, as it is the case for organelles such as ER and peroxisomes (Sawaya et al 2014; Schonherr et al 2015; Tsutsui et al 2015). Therefore, the main information available is on ultrastructure.…”

“…Further, they are found under pathologic conditions, for instance in cancer (Dogan et al 2012) or ischaemia (Hanzlikova and Schiaffino 1977). Crystals have been described in almost all parts of the cell: in metabolic active cell compartments as the endoplasmic reticulum (Hasegawa et al 2011; Jones et al 1999; Koopmann et al 2012), secretory vesicles (Santos 1966) and the cytoplasm (Kozina et al 2011; Schonherr et al 2015) but also at sites of degradation, the lysosomes (Tsutsui et al 2015). Additionally, crystals may appear in mitochondria (Farrants et al 1988), peroxisomes (Schonherr et al 2015) and the nucleus (Gouranton and Thomas 1974).…”

“…Many crystals are very small, rare or unpredictable in their frequency; some even appear as single observations (Klepal et al 2010; Silvestro and Chapman 2004). There are, however, examples of adequate descriptions of crystals in bacteria, in cases with less challenging methodological conditions (Sawaya et al 2014) or in which crystals could be artificially induced, for example via genetic manipulation (Sawaya et al 2014; Schonherr et al 2015; Thamwiriyasati et al 2010; Tsutsui et al 2015) or by means of free-electron laser and serial femtosecond crystallography (Sawaya et al 2014). …”

Protein phase separation and determinants of in cell crystallization

“…Further, in cellulo crystallization has also been associated with several diseases like cataract, hemoglobin C diseases, formation of Charcot‐Leyden crystals (CLCs), Reinke's crystals or mitochondrial myopathies, and more recently in cellulo crystallization was observed as a result of heterologous overexpression of genes in cell lines of bacteria, insect cells, yeast, CHO (Chinese hamster ovary) or HEK (human embryonic kidney) cells . Mostly, these protein crystals were located in different organelles (mitochondria, peroxisomes, lysosomes, or endoplasmic reticulum), as shown in Table but sometimes also within the cytosol or even in the nucleus, as shown in Figure and Table .…”