Liquid-Liquid Phase Separation Phenomenon on Protein Sorting Within Chloroplasts

In higher plants, chloroplasts are vital organelles possessing highly complex compartmentalization. As most chloroplast-located proteins are encoded in the nucleus and synthesized in the cytosol, the correct sorting of these proteins to appropriate compartments is critical for the proper functions of chloroplasts as well as plant survival. Nuclear-encoded chloroplast proteins are imported into stroma and further sorted to distinct compartments via different pathways. The proteins predicted to be sorted to the thylakoid lumen by the chloroplast twin arginine transport (cpTAT) pathway are shown to be facilitated by STT1/2 driven liquid-liquid phase separation (LLPS). Liquid-liquid phase separation is a novel mechanism to facilitate the formation of membrane-less sub-cellular compartments and accelerate biochemical reactions temporally and spatially. In this review, we introduce the sorting mechanisms within chloroplasts, and briefly summarize the properties and significance of LLPS, with an emphasis on the novel function of LLPS in the sorting of cpTAT substrate proteins. We conclude with perspectives for the future research on chloroplast protein sorting and targeting mechanisms.

A Role for Lipids in Protein Sorting?

Lipid and protein diversity provides structural and functional identity to the membrane compartments that define the eukaryotic cell. This compositional heterogeneity is maintained by the secretory pathway, which feeds newly synthesized proteins and lipids to the endomembrane systems. The precise sorting of lipids and proteins through the pathway guarantees the achievement of their correct delivery. Although proteins have been shown to be key for sorting mechanisms, whether and how lipids contribute to this process is still an open discussion. Our laboratory, in collaboration with other groups, has recently addressed the long-postulated role of membrane lipids in protein sorting in the secretory pathway, by investigating in yeast how a special class of lipid-linked cell surface proteins are differentially exported from the endoplasmic reticulum. Here we comment on this interdisciplinary study that highlights the role of lipid diversity and the importance of protein-lipid interactions in sorting processes at the cell membrane.

Coding Mutations in Vacuolar Protein-Sorting 4 AAA+ ATPase Endosomal Sorting Complexes Required for Transport Protein Homologs Underlie bc-2 and New bc-4 Gene Conferring Resistance to Bean Common Mosaic Virus in Common Bean

Bean common mosaic virus (BCMV) is a major disease in common bean (Phaseolus vulgaris L.). Host plant resistance is the most effective strategy to minimize crop damage against BCMV and the related Bean common mosaic necrosis virus (BCMNV). To facilitate breeding for resistance, we sought to identify candidate genes and develop markers for the bc-2 gene and the unknown gene with which it interacts. Genome-wide association study (GWAS) of the Durango Diversity Panel (DDP) identified a peak region for bc-2 on chromosome Pv11. Haplotype mapping narrowed the bc-2 genomic interval and identified Phvul.011G092700, a vacuolar protein-sorting 4 (Vps4) AAA+ ATPase endosomal sorting complexes required for transport (ESCRT) protein, as the bc-2 candidate gene. The race Durango Phvul.011G092700 gene model, bc-2[UI111], contains a 10-kb deletion, while the race Mesoamerican bc-2[Robust] consists of a single nucleotide polymorphism (SNP) deletion. Each mutation introduces a premature stop codon, and they exhibit the same interaction with the pathogroups (PGs) tested. Phvul.005G125100, another Vps4 AAA+ ATPase ESCRT protein, was identified as the candidate gene for the new recessive bc-4 gene, and the recessive allele is likely an amino acid substitution in the microtubule interacting and transport (MIT) domain. The two Vps4 AAA+ ATPase ESCRT proteins exhibit high similarity to the Zym Cucsa.385040 candidate gene associated with recessive resistance to Zucchini yellow mosaic virus in cucumber. bc-2 alone has no resistance effect but, when combined with bc-4, provides resistance to BCMV (except PG-V) but not BCMNV, and, when combined with bc-ud, provides resistance to BCMV (except BCMV PG-VII) and BCMNV. So instead of different resistance alleles (i.e., bc-2 and bc-22), there is only bc-2 with a differential reaction based on whether it is combined with bc-4 or bc-ud, which are tightly linked in repulsion. The new tools and enhanced understanding of this host-virus pathogen interaction will facilitate breeding common beans for resistance to BCMV and BCMNV.

The VINE complex is a VPS9–domain GEF–containing SNX–BAR coat involved in endosomal sorting

Membrane trafficking pathways perform important roles in establishing and maintaining the endolysosomal network. Retrograde protein sorting from the endosome is promoted by conserved SNX–BAR–containing coat complexes including retromer which enrich cargo at tubular microdomains and generate transport carriers. In metazoans, retromer cooperates with VARP, a conserved VPS9–domain GEF, to direct an endosomal recycling pathway. The function of the yeast VARP homolog Vrl1 has been overlooked due an inactivating mutation in commonly studied strains. Here, we demonstrate that Vrl1 has features of a SNX–BAR coat protein and forms an obligate complex with Vin1, the paralog of the retromer SNX–BAR protein Vps5. Unique features in the Vin1 N–terminus allow Vrl1 to distinguish it from Vps5, thereby forming what we have named the VINE complex. VINE occupies endosomal tubules and promotes the delivery of a conserved mannose 6–phosphate receptor–like protein to the vacuolar membrane. In addition to sorting functions, membrane recruitment by Vin1 is essential for Vrl1 GEF activity, suggesting that VINE is a multifunctional coat complex that regulates trafficking and signaling events at the endosome.

Subunit exchange among endolysosomal tethering complexes is linked to contact site formation at the vacuole

The hexameric HOPS (homotypic fusion and protein sorting) complex is a conserved tethering complex at the lysosome-like vacuole, where it mediates tethering and promotes all fusion events involving this organelle. The Vps39 subunit of this complex also engages in a membrane contact site between the vacuole and the mitochondria, called vCLAMP. Additionally, four subunits of HOPS are also part of the endosomal CORVET tethering complex. Here, we analyzed the partition of HOPS and CORVET subunits between the different complexes by tracing their localization and function. We find that Vps39 has a specific role in vCLAMP formation beyond tethering, and that vCLAMPs and HOPS compete for the same pool of Vps39. In agreement, we find that the CORVET subunit Vps3 can take the position of Vps39 in HOPS. This endogenous pool of a Vps3-hybrid complex is affected by Vps3 or Vps39 levels, suggesting that HOPS and CORVET assembly is dynamic. Our data shed light on how individual subunits of tethering complexes such as Vps39 can participate in other functions, while maintaining the remaining subcomplex available for its function in tethering and fusion.

VPS35 Downregulation Alters Degradation Pathways in Neuronal Cells

Background: The vacuolar protein sorting 35 (VPS35) is the main component of the retromer recognition core complex system which regulates intracellular cargo protein sorting and trafficking. Downregulation of VPS35 has been linked to the pathogenesis of neurodegenerative disorders such Alzheimer’s and Parkinson’s diseases via endosome dysregulation. Objective: Here we show that the genetic manipulation of VPS35 affects intracellular degradation pathways. Methods: A neuronal cell line expressing human APP Swedish mutant was used. VPS35 silencing was performed treating cells with VPS35 siRNA or Ctr siRNA for 72 h. Results: Downregulation of VPS35 was associated with alteration of autophagy flux and intracellular accumulation of acidic and ubiquitinated aggregates suggesting that dysfunction of the retromer recognition core leads to a significant alteration in both pathways. Conclusion: Taken together, our data demonstrate that besides cargo sorting and trafficking, VPS35 by supporting the integral function of the retromer complex system plays an important role also as a critical regulator of intracellular degradation pathways.

Protein Sorting in Plasmodium Falciparum

Plasmodium falciparum is a unicellular eukaryote with a very polarized secretory system composed of micronemes rhoptries and dense granules that are required for host cell invasion. P. falciparum, like its relative T. gondii, uses the endolysosomal system to produce the secretory organelles and to ingest host cell proteins. The parasite also has an apicoplast, a secondary endosymbiotic organelle, which depends on vesicular trafficking for appropriate incorporation of nuclear-encoded proteins into the apicoplast. Recently, the central molecules responsible for sorting and trafficking in P. falciparum and T. gondii have been characterized. From these studies, it is now evident that P. falciparum has repurposed the molecules of the endosomal system to the secretory pathway. Additionally, the sorting and vesicular trafficking mechanism seem to be conserved among apicomplexans. This review described the most recent findings on the molecular mechanisms of protein sorting and vesicular trafficking in P. falciparum and revealed that P. falciparum has an amazing secretory machinery that has been cleverly modified to its intracellular lifestyle.