Learning cellular sorting pathways using protein interactions and sequence motifs

Proper subcellular localization is critical for proteins to perform their roles in cellular functions. Proteins are transported by different cellular sorting pathways, some of which take a protein through several intermediate locations until reaching its final destination. The pathway a protein is transported through is determined by carrier proteins that bind to specific sequence motifs. In this article, we present a new method that integrates protein interaction and sequence motif data to model how proteins are sorted through these sorting pathways. We use a hidden Markov model (HMM) to represent protein sorting pathways. The model is able to determine intermediate sorting states and to assign carrier proteins and motifs to the sorting pathways. In simulation studies, we show that the method can accurately recover an underlying sorting model. Using data for yeast, we show that our model leads to accurate prediction of subcellular localization. We also show that the pathways learned by our model recover many known sorting pathways and correctly assign proteins to the path they utilize. The learned model identified new pathways and their putative carriers and motifs and these may represent novel protein sorting mechanisms. Supplementary results and software implementation are available from http://murphylab.web.cmu.edu/software/2010_RECOMB_pathways/.     

Cis-elements of protein transport to the plant vacuoles

Vacuolar proteins are synthesized and translocated into the endoplasmic reticulum and transported to the vacuoles through the secretory pathway. Three different types of vacuolar sorting signals have been identified, carried by N- or C-terminal propeptides or internal sequences. These signals are needed to target proteins to the different types of vacuoles that can coexist in a single plant cell. A conserved motif (NPIXL or NPIR) was identified within N-terminal propeptides, but can also function in a C-terminal propeptide and targets proteins in a receptor-mediated manner to a lytic vacuole. Binding to a family of putative sorting receptors for sequence-specific vacuolar sorting signals has been used as an assay to identify further peptides with other binding motifs. No motif was found in C-terminal sorting sequences, which need an accessible terminus, suggesting that they are recognized from the end by a still unknown receptor. The phosphatidylinositol kinase inhibitor wortmannin differentially affects sorting mediated by these two sorting sequences, suggesting different sorting mechanisms. Less is known about sorting mediated by internal protein sequences, which do not contain the conserved motif identified in N-terminal propeptides and by function by aggregation, leading to transport by coat-less dense vesicles to protein storage vacuoles. Even less is known about the sorting of tonoplast proteins, for which several sorting systems will also be needed.     

N-glycans mediate apical recycling of the sialomucin endolyn in polarized MDCK cells

Apical and basolateral proteins are maintained within distinct membrane subdomains in polarized epithelial cells by biosynthetic and postendocytic sorting processes. Sorting of basolateral proteins in these processes has been well studied; however, the sorting signals and mechanisms that direct proteins to the apical surface are less well understood. We previously demonstrated that an N-glycan-dependent sorting signal directs the sialomucin endolyn to the apical surface in polarized Madin-Darby canine kidney cells. Terminal processing of a subset of endolyn's N-glycans is key for polarized biosynthetic delivery to the apical membrane. Endolyn is subsequently internalized, and via a cytoplasmic tyrosine-based sorting motif is targeted to lysosomes from where it constitutively cycles to the cell surface. Here, we examine the polarized sorting of endolyn along the postendocytic pathway in polarized cells. Our results suggest that similar N-glycan sorting determinants are required for apical delivery of endolyn along both the biosynthetic and the postendocytic pathways.     

Lack of a vacuolar sorting receptor leads to non-specific missorting of soluble vacuolar proteins in Arabidopsis seeds

The plant vacuolar sorting receptor (VSR) binds proteins carrying vacuolar sorting signals (VSS) of the 'sequence-specific' type (ssVSS) but not the C-terminal, hydrophobic sorting signals (ctVSS). Seeds of Arabidopsis mutants lacking the major VSR isoform, AtVSR1, secrete a proportion of the proteins destined to storage vacuoles. The sorting signals for these proteins are not well defined, but they do not seem to be of the ssVSS type. Here, we tested whether absence of VSR1 in seeds leads to secretion of reporter proteins carrying ssVSS but not ctVSS. Our results show that reporters carrying either ssVSS or ctVSS are equally secreted in the absence of VSR1. We discuss our findings in relation to the current model for vacuolar sorting.     

Ubiquitin-dependent sorting of integral membrane proteins for degradation in lysosomes

The pathways that deliver newly synthesized proteins that reside in lysosomes are well understood on comparison with our knowledge of how integral membrane proteins are sorted and delivered to the lysosome for degradation. Many membrane proteins are sorted to lysosomes following ubiquitination, which provides a sorting signal that can operate for sorting at the TGN (trans-Golgi network), at the plasma membrane or at the endosome for delivery into lumenal vesicles. Candidate multicomponent machines that can potentially move ubiquitinated integral membrane cargo proteins have been identified, but much work is still required to ascertain which of these candidates directly recognize ubiquitinated cargo and what they do with cargo after recognition. In the case of the machinery required for sorting into the lumenal vesicles of endosomes, other functions have also been determined including a link between sorting and movement of endosomes along microtubules.     

Sorting pathways of mitochondrial inner membrane proteins

Two distinct pathways of sorting and assembly of nuclear-encoded mitochondrial inner membrane proteins are described. In the first pathway, precursor proteins that carry amino-terminal targeting signals are initially translocated via contact sites between both mitochondrial membranes into the mitochondrial matrix. They become proteolytically processed, interact with the 60-kDa heat-shock protein hsp60 in the matrix and are retranslocated to the inner membrane. The sorting of subunit 9 of Neurospora crassa F0-ATPase has been studied as an example. F0 subunit 9 belongs to that class of nuclear-encoded mitochondrial proteins which are evolutionarily derived from a prokaryotic ancestor according to the endosymbiont hypothesis. We suggest that after import into mitochondria, these proteins follow the ancestral sorting and assembly pathways established in prokaryotes (conservative sorting). On the other hand, ADP/ATP carrier was found not to require interaction with hsp60 for import and assembly. This agrees with previous findings that the ADP/ATP carrier possesses non-amino-terminal targeting signals and uses a different import receptor to other mitochondrial precursor proteins. It is proposed that the ADP/ATP carrier represents a class of mitochondrial inner membrane proteins which do not have a prokaryotic equivalent and thus appear to follow a non-conservative sorting pathway.     

A deubiquitinating enzyme UBPY regulates the level of protein ubiquitination on endosomes

Monoubiquitination of endocytosed cell surface receptors serves as a sorting signal for their trafficking from endosomes to lysosomes. The sorting of ubiquitinated proteins is executed by concerted actions of class E vacuolar protein sorting (Vps) proteins. Some proteins in the sorting machinery undergo monoubiquitination, suggesting that their functions are also regulated by ubiquitination. The Hrs-STAM complex, a class E Vps protein complex essential for the initial step of the sorting pathway, binds two deubiquitinating enzymes, UBPY and AMSH. Here we examined the effects of inactivating UBPY on protein ubiquitination at endosomes. Overexpression of a catalytically inactive UBPY mutant or depletion of UBPY by RNA interference resulted in the accumulation of ubiquitinated proteins on morphologically aberrant endosomes. Electron microscopy showed that they are aggregates of multivesicular endosomes. Among the sorting machinery proteins that undergo ubiquitination, Eps15 was monoubiquitinated at an elevated level in UBPY-inactivated cells. UBPY also deubiquitinated Eps15 in vitro, suggesting that Eps15 is a cellular substrate for UBPY. Furthermore, inactivation of UBPY caused the accumulation of Eps15 on the endosomal aggregates. These results suggest that UBPY regulates the level of protein ubiquitination on endosomes, which is required for maintaining the morphology of the organelle.     

Sorting and targeting of melanosomal membrane proteins: signals, pathways, and mechanisms

Newly synthesized melanosomal proteins, like many other cellular proteins, traverse through a series of intracellular compartments en route to melanosomes. Entry and exit of proteins through these compartments is orchestrated by cellular sorting machinery that recognize specific sorting signals. Melanosomal membrane proteins begin their intracellular journey upon co-translational importation into the endoplasmic reticulum (ER). The biosynthetic output of tyrosinase, the key melanogenic enzyme, appears to be regulated by quality-control events at the ER, the 'port of entry' to the secretory pathway. Following maturation in the ER and through the Golgi, the sorting of these proteins in the trans-Golgi network for intracellular retention and transport along endosome/lysosome pathway requires cytoplasmically exposed signals. A di-leucine motif, present in the cytoplasmic tails of most melanosomal proteins, and its interaction with adaptor protein (AP) complexes, specifically AP-3, are critical for these events. Defects in sorting signals and the cytosolic components that interact with these signals result in a number of murine coat color phenotypes and cause human pigmentary disorders. Thus, missense or frame-shift mutations that produce truncated tyrosinase lacking the melanosomal sorting signal(s) appear to be responsible for murine platinum coat color phenotypes and a proportion of human oculocutaneous albinism-1; mutations in AP-3 appear to be responsible for the mocha phenotype in mice and Hermansky-Pudlak-like syndrome in man. Additional signals and sorting steps downstream of AP-3 appear to be required for endosomal sorting and targeting proteins to melanosomes. Signals and mechanisms that sequester melanosomal proteins from endosomes/lysosomes are not understood. Potential candidates that mediate such processes include proteins encoded by lyst and pallid genes. The common occurrence of abnormalities in melanosomes in many storage-pool disorders suggests that melanocytes utilize signals, pathways, and mechanisms shared by other proteins and cell types to assemble a number of specialized proteins and produce unique cell-type-specific organelles.     

Mechanisms regulating the sorting of soluble lysosomal proteins

Lysosomes are key regulators of many fundamental cellular processes such as metabolism, autophagy, immune response, cell signalling and plasma membrane repair. These highly dynamic organelles are composed of various membrane and soluble proteins, which are essential for their proper functioning. The soluble proteins include numerous proteases, glycosidases and other hydrolases, along with activators, required for catabolism. The correct sorting of soluble lysosomal proteins is crucial to ensure the proper functioning of lysosomes and is achieved through the coordinated effort of many sorting receptors, resident ER and Golgi proteins, and several cytosolic components. Mutations in a number of proteins involved in sorting soluble proteins to lysosomes result in human disease. These can range from rare diseases such as lysosome storage disorders, to more prevalent ones, such as Alzheimer’s disease, Parkinson’s disease and others, including rare neurodegenerative diseases that affect children. In this review, we discuss the mechanisms that regulate the sorting of soluble proteins to lysosomes and highlight the effects of mutations in this pathway that cause human disease. More precisely, we will review the route taken by soluble lysosomal proteins from their translation into the ER, their maturation along the Golgi apparatus, and sorting at the trans-Golgi network. We will also highlight the effects of mutations in this pathway that cause human disease.     

Basic mechanisms of secretion: sorting into the regulated secretory pathway.

Targeting proteins to their correct cellular location is crucial for their biological function. In neuroendocrine cells, proteins can be secreted by either the constitutive or the regulated secretory pathways but the mechanism(s) whereby proteins are sorted into either pathway is unclear. In this review we discuss the possibility that sorting is either an active process occurring at the level of the trans-Golgi network, or that sorting occurs passively in the immature granules, The possible involvement of protein-lipid interactions in the sorting process is also raised.     

Direct apical sorting of rat liver dipeptidylpeptidase IV expressed in Madin-Darby canine kidney cells.

In Madin-Darby canine kidney (MDCK) cells, apical and basolateral membrane proteins are segregated from each other in the trans-Golgi network (TGN) and are transported to the appropriate membrane domain via separate vesicle populations. In hepatocytes, however, all plasma membrane proteins are delivered basolaterally. Apical proteins are then selectively retrieved and reach the apical surface by transcytosis. The sorting of apical proteins in different cell types may be the result of differences in the cellular sorting machinery, or alternatively, due to expression of cell-specific sorting signals on the proteins themselves. To test this directly, we have stably expressed cDNA encoding an apical protein from rat liver, dipeptidylpeptidase IV (DPPIV), in MDCK cells. We found that approximately 90% of the exogenous DPPIV is expressed on the apical cell surface at steady state. Furthermore, we demonstrate that this distribution is primarily due to vectorial transport from the TGN to the apical plasma membrane. The small pool of mis-sorted DPPIV that appears basolaterally is slowly endocytosed (t1/2 approximately 60 min) and is subsequently transcytosed. These data are consistent with the notion that both hepatocytes and MDCK cells are capable of correctly sorting rat liver DPPIV, but that this sorting occurs at different sites in the two cell types.     

Insertion of bitopic membrane proteins into the inner membrane of mitochondria involves an export step from the matrix

The mitochondrial inner membrane contains a large number of polytopic proteins that are derived from prokaryotic ancestors of mitochondria. Little is known about the intramitochondrial sorting of these proteins. We chose two proteins of known topology as examples to study the pathway of insertion into the inner membrane; Mrs2 and Yta10 are bitopic proteins that expose negatively charged loops of different complexity into the intermembrane space. Here we show that both Mrs2 and Yta10 transiently accumulate as sorting intermediates in the matrix before they integrate into the inner membrane. The sorting pathway of both proteins can be separated into two sequential reactions: (i) import into the matrix and (ii) insertion from the matrix into the inner membrane. The latter process was found to depend on the membrane potential and, in this respect, is similar to the insertion of membrane proteins in bacteria. A comparison of the charge distribution of intermembrane space loops in a variety of mitochondrial inner membrane proteins suggests that this mode of "conservative sorting" might be the typical insertion route for polytopic inner membrane proteins that originated from bacterial ancestors.     

Protein sorting and secretion granule formation in regulated secretory cells

Formation of secretion granules in regulated secretory cells involves packaging a subject of proteins undergoing intracellular transport into specific vesicular carriers that function in stimulus-dependent exocytosis. Recent findings suggest that immature granules are a site of passive sorting, involving condensation of regulated secretory proteins. Proteins that are not condensed are stored to a lesser degree and are enriched in unstimulated, constitutive-like secretion. While these observations have helped to distinguish possible mechanisms of secretory protein sorting, there are only recent hints about the sorting processes that may be required to create the regulated secretory carrier membranes.     

Tetraspan cargo adaptors usher GPI-anchored proteins into multivesicular bodies

Ubiquitinated membrane proteins are sorted into intralumenal endosomal vesicles on their way for degradation in lysosomes. Here we summarize the discovery of the Cos proteins, which work to organize and segregate ubiquitinated cargo prior to its incorporation into intralumenal vesicles of the multivesicular body (MVB). Importantly, cargoes such as GPI-anchored proteins (GPI-APs) that cannot undergo ubiquitination, rely entirely on Cos proteins for sorting into intralumenal vesicles using the same pathway that depends on ESCRTs and ubiquitin ligases that typical polytopic membrane proteins do. Here we show Cos proteins provide functions as not only adaptor proteins for ubiquitin ligases, but also as cargo carriers that can physically usher a variety of other proteins into the MVB pathway. We then discuss the significance of this new sorting model and the broader implications for this cargo adaptor mechanism, whereby yeast Cos proteins, and their likely animal analogs, provide a ubiquitin sorting signal in trans to enable sorting of a membrane protein network into intralumenal vesicles.     

A hepatocyte-specific basolateral membrane protein is targeted to the same domain when expressed in Madin-Darby canine kidney cells

Different mechanisms for polarized sorting of apical and basolateral plasma membrane proteins appear to be operative in different cell types. In hepatocytes, all proteins are first transported to the basolateral surface, where sorting (probably signal-mediated) of apical proteins then takes place. In contrast, in Madin-Darby canine kidney (MDCK) cells, proteins are directly transported from the trans-Golgi network to their appropriate plasma membrane domain. In order to study the differences in the sorting requirements of the two cell types, we have expressed a hepatocyte-specific basolateral membrane protein, the asialoglycoprotein receptor H1, in MDCK cells. H1 was found to be specifically transported to the basolateral domain also in this heterologous system, suggesting that either the same basolateral targeting signal is operative in both cell types or, more likely, that basolateral transport occurs "by default," i.e. without the requirement for a sorting signal.     

Secretory cargo composition affects polarized secretion in MDCK epithelial cells

Polarized epithelial cells secrete proteins at either the apical or basolateral cell surface. A number of non-epithelial secretory proteins also exhibit polarized secretion when they are expressed in polarized epithelial cells but it is difficult to predict where foreign proteins will be secreted in epithelial cells. The question is of interest since secretory epithelia are considered as target tissues for gene therapy protocols that aim to express therapeutic secretory proteins. In the parathyroid gland, parathyroid hormone is processed by furin and co-stored with chromogranin A in secretory granules. To test the secretion of these proteins in epithelial cells, they were expressed in MDCK cells. Chromogranin A and a secreted form of furin were secreted apically while parathyroid hormone was secreted 60% basolaterally. However, in the presence of chromogranin A, the secretion of parathyroid hormone was 65% apical, suggesting that chromogranin can act as a “sorting escort” (sorting chaperone) for parathyroid hormone. Conversely, apically secreted furin did not affect the sorting of parathyroid hormone. The apical secretion of chromogranin A was dependent on cholesterol, suggesting that this protein uses an established cellular sorting mechanism for apical secretion. However, this sorting does not involve the N-terminal membrane-binding domain of chromogranin A. These results suggest that foreign secretory proteins can be used as “sorting escorts” to direct secretory proteins to the apical secretory pathway without altering the primary structure of the secreted protein. Such a system may be of use in the targeted expression of secretory proteins from epithelial cells. David V. Cohn—Deceased.     

The ER v-SNAREs are required for GPI-anchored protein sorting from other secretory proteins upon exit from the ER

Glycosylphosphatidylinositol (GPI)-anchored proteins exit the ER in distinct vesicles from other secretory proteins, and this sorting event requires the Rab GTPase Ypt1p, tethering factors Uso1p, and the conserved oligomeric Golgi complex. Here we show that proper sorting depended on the vSNAREs, Bos1p, Bet1p, and Sec22p. However, the t-SNARE Sed5p was not required for protein sorting upon ER exit. Moreover, the sorting defect observed in vitro with bos1-1 extracts was also observed in vivo and was visualized by EM. Finally, transport and maturation of the GPI-anchored protein Gas1p was specifically affected in a bos1-1 mutant at semirestrictive temperature. Therefore, we propose that v-SNAREs are part of the cargo protein sorting machinery upon exit from the ER and that a correct sorting process is necessary for proper maturation of GPI-anchored proteins.     

Vps4 regulates a subset of protein interactions at the multivesicular endosome

During endocytic transport, specific integral membrane proteins are sorted into intraluminal vesicles that bud from the limiting membrane of the endosome. This process, known as multivesicular body (MVB) sorting, is important for several important biological processes. Moreover, components of the MVB sorting machinery are implicated in virus budding. During MVB sorting, a cargo protein recruits components of the MVB sorting machinery from cytoplasmic pools and these sequentially assemble on the endosome. Disassembly of these proteins and recycling into the cytoplasm is critical for MVB sorting. Vacuolar protein sorting 4 (Vps4) is an AAA (ATPase associated with a variety of cellular activities) ATPase which has been proposed to play a critical role in disassembly of the MVB sorting machinery. However, the mechanism by which it disassembles the complex is not clear. Vps4 contains an N-terminal microtubule interacting and trafficking (MIT) domain, which has previously been shown to be required for recruitment to endosomes, and a single AAA ATPase domain, the activity of which is required for Vps4 function. In this study we have systematically characterized the interaction of Vps4 with other components of the MVB sorting machinery. We demonstrate that Vps4 interacts directly with Vps2 and Bro1. We also show that a subset of Vps4 interactions is regulated by ATP hydrolysis, and one interaction is regulated by ATP binding. Finally, we show that most proteins interact with the Vps4 MIT domain. Our studies indicate that the MIT domain has a dual role in substrate binding and recruitment to endosomes and indicate that Vps4 disassembles the MVB sorting machinery by direct effects on multiple proteins.     

A new class of lysosomal/vacuolar protein sorting signals

A number of inherited lysosomal diseases are known to result from missorting of lysosomal proteins. Considerable attention has been directed toward an understanding of this sorting pathway, and it has become apparent that different mechanisms are used for the sorting of lysosomal membrane and soluble proteins. Protein sorting to the yeast vacuole/lysosome provides a simple model system to study this process. We have mapped the first sorting signal in a vacuolar membrane protein, repressible alkaline phosphatase, and have shown it to be both necessary and sufficient for vacuolar delivery of this enzyme. The sorting information is confined to the transmembrane and cytoplasmic tail region of this type II integral membrane protein. The location of this sorting signal provides an explanation for some of the differences observed between membrane and soluble vacuolar protein sorting.     

The Vps27p Hse1p complex binds ubiquitin and mediates endosomal protein sorting

Membrane proteins that are degraded in the vacuole of Saccharomyces cerevisiae are sorted into discrete intralumenal vesicles, analogous to the internal membranes of multi-vesiculated bodies (MVBs). Recently, it has shown that the attachment of ubiquitin (Ub) mediates sorting into lumenal membranes. We describe a complex of Vps27p and Hse1p that localizes to endosomal compartments and is required for the recycling of Golgi proteins, formation of lumenal membranes and sorting of ubiquitinated proteins into those membranes. The Vps27p-Hse1p complex binds to Ub and requires multiple Ub Interaction Motifs (UIMs). Mutation of these motifs results in specific defects in the sorting of ubiquitinated proteins into the vacuolar lumen. However, the recycling of Golgi proteins and the generation of lumenal membranes proceeds normally in Delta UIM mutants. These data support a model in which the Vps27p-Hse1p complex has multiple functions at the endosome, one of which is as a sorting receptor for ubiquitinated membrane proteins destined for degradation.