As an essential organelle in the cell, the lysosome is responsible for digestion and recycling of intracellular components, storage of nutrients, and pH homeostasis. The lysosome is enclosed by a special membrane to maintain its integrity, and nutrients are transported across the membrane by numerous transporters. Despite their importance in maintaining nutrient homeostasis and regulating signaling pathways, little is known about how lysosomal membrane protein lifetimes are regulated. We identified a yeast vacuolar amino acid transporter, Ypq1, that is selectively sorted and degraded in the vacuolar lumen following lysine withdrawal. This selective degradation process requires a vacuole anchored ubiquitin ligase (VAcUL-1) complex composed of Rsp5 and Ssh4. We propose that after ubiquitination, Ypq1 is selectively sorted into an intermediate compartment. The ESCRT machinery is then recruited to sort the ubiquitinated Ypq1 into intraluminal vesicles (ILVs). Finally, the compartment fuses with the vacuole and delivers ILVs into the lumen for degradation.

The chloroplast envelope plays critical roles in the synthesis and regulated transport of key metabolites, including intermediates in photosynthesis and lipid metabolism. Despite this importance, the biogenesis of the envelope membranes has not been investigated in detail. To identify the determinants of protein targeting to the inner envelope membrane (IM), we investigated the targeting of the nucleus-encoded integral IM protein, atTic40. We found that pre-atTic40 is imported into chloroplasts and processed to an intermediate size (int-atTic40) before insertion into the IM. Int-atTic40 is soluble and inserts into the IM from the internal stromal compartment. We also show that atTic40 and a second IM protein, atTic110, can target and insert into isolated IM vesicles in vitro. Collectively, our experiments are consistent with a “postimport” mechanism in which the IM proteins are first imported from the cytoplasm and subsequently inserted into the IM from the stroma.

The lysosome plays an important role in maintaining cellular nutrient homeostasis. Regulation of nutrient storage can occur by the ubiquitination of certain transporters that are then sorted into the lysosome lumen for degradation. To better understand the underlying mechanism of this process, we performed genetic screens to identify components of the sorting machinery required for vacuole membrane protein degradation. These screens uncovered genes that encode a ubiquitin ligase complex, components of the PtdIns 3-kinase complex, and the ESCRT machinery. We developed a novel ubiquitination system, Rapamycin-Induced Degradation (RapiDeg), to test the sorting defects caused by these mutants. These tests revealed that ubiquitinated vacuole membrane proteins recruit ESCRTs to the vacuole surface, where they mediate cargo sorting and direct cargo delivery into the vacuole lumen. Our findings demonstrate that the ESCRTs can function at both the late endosome and the vacuole membrane to mediate cargo sorting and intra-luminal vesicle formation.DOI: http://dx.doi.org/10.7554/eLife.26403.001

Chloroplast heat shock protein 90 (Hsp90C) represents a highly conserved subfamily of the Hsp90 family of molecular chaperones whose function has not been defined. We identified Hsp90C as a component that interacts with import intermediates of nuclear-encoded preproteins during posttranslational import into isolated chloroplasts. Hsp90C was specifically coprecipitated with a complex of protein import components, including Tic110, Tic40, Toc75, Tic22, and the stromal chaperones, Hsp93 and Hsp70. Radicicol, an inhibitor of Hsp90 ATPase activity, reversibly inhibited the import of a variety of preproteins during translocation across the inner envelope membrane, indicating that Hsp90C functions in membrane translocation into the organelle. Hsp90C is encoded by a single gene in Arabidopsis thaliana, and insertion mutations in the Hsp90C gene are embryo lethal, indicating an essential function for the chaperone in plant viability. On the basis of these results, we propose that Hsp90C functions within a chaperone complex in the chloroplast stroma to facilitate membrane translocation during protein import into the organelle.

VAcUL-1 and Dsc, two different E3 ubiquitin ligase complexes coexist on the vacuole membrane to regulate different subsets of membrane proteins via the vReD pathway in response to different environmental cues.

PI(3,5)P2 is directly linked to generation and remodeling of the cortical actin array by formin during polarized cell growth in plants.

Retromer is an evolutionarily conserved protein complex, which sorts functionally diverse membrane proteins into recycling tubules/vesicles from the endosome. Many of the identified cargos possess a recycling signal sequence defined as ØX[L/M/V], where Ø is F/Y/W. However, this sequence is present in almost all proteins encoded in the genome. Also, several identified recycling sequences do not follow this rule. How then does retromer precisely select its cargos? Here, we reveal that an additional motif is also required for cargo retrieval. The two distinct motifs form a bipartite recycling signal recognized by the retromer subunits, Vps26 and Vps35. Strikingly, Vps26 utilizes different binding sites depending on the cargo, allowing retromer to recycle different membrane proteins. Thus, retromer interacts with cargos in a more complex manner than previously thought, which facilitates precise cargo recognition.

The inner envelope membrane (IEM) of the chloroplast plays key roles in controlling metabolite transport between the organelle and cytoplasm and is a major site of lipid and membrane synthesis within the organelle. IEM biogenesis requires the import and integration of nucleus-encoded membrane proteins. Previous reports have led to the conclusion that membrane proteins are inserted into the IEM during protein import from the cytoplasm via a stop-transfer mechanism or are completely imported into the stroma and then inserted into the IEM in a post-import mechanism. In this study, we examined the determinants for each pathway by comparing the targeting of APG1 (albino or pale green mutant 1), an example of a stoptransfer substrate, and atTic40, an example of a post-import substrate. We show that the APG1 transmembrane domain is sufficient to direct stop-transfer insertion. The APG1 transmembrane domain also functions as a topology determinant. We also show that the ability of the post-import signals within atTic40 to target proteins to the IEM is dependent upon their context within the full protein sequence. In the incorrect context, the atTic40 signals can behave as stop-transfer signals or fail to target fusion proteins to the IEM. These data suggest that the post-import pathway signals are complex and have evolved to avoid stop-transfer insertion.The chloroplast is a structurally complex organelle that performs diverse metabolic functions (1). It is composed of six distinguishable compartments, including three membranes (the outer envelope membrane, the inner envelope membrane, and the thylakoid membrane) and three aqueous and hydrophilic compartments (the intermembrane space (IMS), 2 located between the two envelope membranes, the stroma, and the thylakoid lumen) (2). These compartments are dependent upon the import and proper suborganellar targeting of several thousand nucleus-encoded proteins (3).The translocon of the outer envelope membrane of chloroplasts (TOC) and translocon of the inner envelope membrane of chloroplasts (TIC) complexes interact to mediate the import of the vast majority of nucleus-encoded proteins from the cytoplasm into the chloroplast stroma (4, 5). The TOC-TIC system recognizes the intrinsic N-terminal transit peptides of newly synthesized preproteins and catalyzes translocation across both envelope membranes simultaneously (2, 6, 7). In the case of thylakoid-targeted proteins, the targeting signals are bipartite. In addition to a transit peptide, thylakoid proteins contain secondary signals that target them from the stroma to the thylakoid. These processes occur in two independent steps (8, 9). The targeting of proteins to the thylakoid resembles protein export in bacteria because they utilize similar mechanisms (10, 11). For that reason, these pathways are referred to as "conservative sorting."Much less is known about the mechanisms of protein targeting and insertion at the chloroplast envelope membranes. This is surprising, considering the central role of the envelope in cellular me...