Palmitoylation controls recycling in lysosomal sorting and trafficking

For the efficient trafficking of lysosomal proteins, the cationic-dependent and -independent mannose 6-phosphate receptors and sortilin must bind cargo in the Golgi apparatus, be packaged into clathrin-coated trafficking vesicles and traffic to the endosomes. Once in the endosomes, the receptors release their cargo into the endosomal lumen and recycle back to the Golgi for another round of trafficking, a process that requires retromer. In this study, we demonstrate that palmitoylation is required for the efficient retrograde trafficking of sortilin, and the cationic-independent mannose 6-phosphate as palmitoylation-deficient receptors remain trapped in the endosomes. Importantly, we also show that palmitoylation is required for receptor interaction with retromer as nonpalmitoylated receptor did not interact with retromer. In addition, we have identified DHHC-15 as the palmitoyltransferase responsible for this modification. In summary, we have shown the functional significance of palmitoylation in lysosomal receptor sorting and trafficking.     

Intracellular trafficking of lysosomal membrane proteins

Lysosomes are the site of degradation of obsolete intracellular material during autophagy and of extracellular macromolecules following endocytosis and phagocytosis. The membrane of lysosomes and late endosomes is enriched in highly glycosylated transmembrane proteins of largely unknown function. Significant progress has been made in recent years towards elucidating the pathways by which these lysosomal membrane proteins are delivered to late endosomes and lysosomes. While some lysosomal membrane proteins follow the constitutive secretory pathway and reach lysosomes indirectly via the cell surface and endocytosis, others exit the trans-Golgi network in clathrin-coated vesicles for direct delivery to endosomes and lysosomes. Sorting from the Golgi or the plasma membrane into the endosomal system is mediated by signals encoded by the short cytosolic domain of these proteins. This review will discuss the role of lysosomal membrane proteins in the biogenesis of the late endosomal and lysosomal membranes, with particular emphasis on the structural features and molecular mechanisms underlying the intracellular trafficking of these proteins.     

AP-1 and AP-3 mediate sorting of melanosomal and lysosomal membrane proteins into distinct post-Golgi trafficking pathways

The adaptor complexes AP-1 and AP-3 are localized to endosomes and/or the trans Golgi network (TGN). Because of limitations in analysing intracellular adaptor function directly, their site of function is a matter of ongoing uncertainty. To overcome this problem and to analyse adaptor sorting at the TGN, we reconstituted vesicle formation from Golgi/TGN-enriched membranes in a novel in vitro budding assay. Melanocytes were metabolically labelled followed by a 19°C temperature block to accumulate newly synthesized proteins in Golgi membranes, which were then enriched by subcellular fractionation and used as donor membranes for vesicle formation in vitro . The incorporation of the melanosomal proteins tyrosinase and tyrosinase-related protein 1 (TRP-1) as well as Lamp-1 and 46 kDa mannose-6-phosphate receptor (MPR46) into Golgi/TGN-derived vesicles was temperature, nucleotide, cytosol, ADP ribosylation factor 1 and adaptor dependent. We show that sorting of TRP-1 and MPR46 was AP-1 dependent, while budding of tyrosinase and Lamp-1 required AP-3. Depletion of clathrin inhibited sorting of all four cargo proteins, suggesting that AP-1 and AP-3 are involved in the formation of distinct types of clathrin-coated vesicles, each of which is characterized by the incorporation of specific cargo membrane proteins.     

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.     

Glycolipid-dependent sorting of melanosomal from lysosomal membrane proteins by lumenal determinants

Melanosomes are lysosome-related organelles that coexist with lysosomes in mammalian pigment cells. Melanosomal and lysosomal membrane proteins share similar sorting signals in their cytoplasmic tail, raising the question how they are segregated. We show that in control melanocytes, the melanosomal enzymes tyrosinase-related protein 1 (Tyrp1) and tyrosinase follow an intracellular Golgi to melanosome pathway, whereas in the absence of glycosphingolipids, they are observed to pass over the cell surface. Unexpectedly, the lysosome-associated membrane protein 1 (LAMP-1) and 2 behaved exactly opposite: they were found to travel through the cell surface in control melanocytes but followed an intracellular pathway in the absence of glycosphingolipids. Chimeric proteins having the cytoplasmic tail of Tyrp1 or tyrosinase were transported like lysosomal proteins, whereas a LAMP-1 construct containing the lumenal domain of Tyrp1 localized to melanosomes. In conclusion, the lumenal domain contains sorting information that guides Tyrp1 and probably tyrosinase to melanosomes by an intracellular route that excludes lysosomal proteins and requires glucosylceramide.     

The Niemann-Pick C proteins and trafficking of cholesterol through the late endosomal/lysosomal system

To maintain proper cellular function, the amount and distribution of cholesterol residing within cellular membranes must be regulated. The principal disorder affecting transport of cholesterol through the late endosomal/lysosomal system and intracellular cholesterol homeostasis is Niemann-Pick type C (NPC) disease. The genes responsible for NPC disease have been identified, and the encoded Niemann-Pick C1 (NPC1) and Niemann-Pick C2 (HE1/NPC2) proteins are currently the subject of intense investigation. This review provides a detailed examination of NPC1 and HE1/NPC2 in regulating the transport of cholesterol through the late endosomal/lysosomal system to other cellular compartments responsible for maintaining intracellular cholesterol homeostasis, and how defective function of these proteins may be responsible for the pathophysiology associated with NPC disease.     

Kinetics of intracellular transport and sorting of lysosomal membrane and plasma membrane proteins

We have used monospecific antisera to two lysosomal membrane glycoproteins, lgp120 and a similar protein, lgp110, to compare the biosynthesis and intracellular transport of lysosomal membrane components, plasma membrane proteins, and lysosomal enzymes. In J774 cells and NRK cells, newly synthesized lysosomal membrane and plasma membrane proteins (the IgG1/IgG2b Fc receptor or influenza virus hemagglutinin) were transported through the Golgi apparatus (defined by acquisition of resistance to endo-beta-N-acetylglucosaminidase H) with the same kinetics (t1/2 = 11-14 min). In addition, immunoelectron microscopy of normal rat kidney cells showed that lgp120 and vesicular stomatitis virus G-protein were present in the same Golgi cisternae demonstrating that lysosomal and plasma membrane proteins were not sorted either before or during transport through the Golgi apparatus. To define the site at which sorting occurred, we compared the kinetics of transport of lysosomal and plasma membrane proteins and a lysosomal enzyme to their respective destinations. Newly synthesized proteins were detected in dense lysosomes (lgp's and beta-glucuronidase) or on the cell surface (Fc receptor or hemagglutinin) after the same lag period (20-25 min), and accumulated at their final destinations with similar kinetics (t1/2 = 30-45 min), suggesting that these two lgp's are not transported to the plasma membrane before reaching lysosomes. This was further supported by measurements of the transport of membrane-bound endocytic markers from the cell surface to lysosomes, which exhibited additional lag periods of 5-15 min and half-times of 1.5-2 h. The time required for transport of newly synthesized plasma membrane proteins to the cell surface, and for the transport of plasma membrane markers from the cell surface to lysosomes would appear too long to account for the rapid transport of lgp's from the Golgi apparatus to lysosomes. Thus, the observed kinetics suggest that lysosomal membrane proteins are sorted from plasma membrane proteins at a post-Golgi intracellular site, possibly the trans Golgi network, before their delivery to lysosomes.     

The lysosomal trafficking of sphingolipid activator proteins (SAPs) is mediated by sortilin

Most soluble lysosomal proteins bind the mannose 6-phosphate receptor (M6P-R) to be sorted to the lysosomes. However, the lysosomes of I-cell disease (ICD) patients, a condition resulting from a mutation in the phosphotransferase that adds mannose 6-phosphate to hydrolases, have near normal levels of several lysosomal proteins, including the sphingolipid activator proteins (SAPs), GM2AP and prosaposin. We tested the hypothesis that SAPs are targeted to the lysosomal compartment via the sortilin receptor. To test this hypothesis, a dominant-negative construct of sortilin and a sortilin small interfering RNA (siRNA) were introduced into COS-7 cells. Our results showed that both the truncated sortilin and the sortilin siRNA block the traffic of GM2AP and prosaposin to the lysosomal compartment. This observation was confirmed by a co-immunoprecipitation, which demonstrated that GM2AP and prosaposin are interactive partners of sortilin. Furthermore, a dominant-negative mutant GGA prevented the trafficking of prosaposin and GM2AP to lysosomes. In conclusion, our results show that the trafficking of SAPs is dependent on sortilin, demonstrating a novel lysosomal trafficking.     

Polarized sorting and trafficking in epithelial cells

The polarized distribution of proteins and lipids at the surface membrane of epithelial cells results in the formation of an apical and a basolateral domain, which are separated by tight junctions. The generation and maintenance of epithelial polarity require elaborate mechanisms that guarantee correct sorting and vectorial delivery of cargo molecules. This dynamic process involves the interaction of sorting signals with sorting machineries and the formation of transport carriers. Here we review the recent advances in the field of polarized sorting in epithelial cells. We especially highlight the role of lipid rafts in apical sorting.     

Overexpression of proteins containing tyrosine- or leucine-based sorting signals affects transferrin receptor trafficking.

Targeting of many transmembrane proteins to post-Golgi compartments is dependent on cytoplasmically exposed sorting signals. The most widely used signals conform to the tyrosine- or the leucine-based motifs. Both types of signals have been implicated in protein localization to the same intracellular compartments, but previous results from both cell-free experiments and studies of transfected cell lines have indicated that the two types of signals interact with separate components of the sorting machinery. We have overexpressed several transmembrane proteins in stably transfected Madin-Darby canine kidney cells using an inducible promoter system. Overexpression of proteins containing tyrosine- or leucine-based sorting signals resulted in reduced internalization of the transferrin receptor, whereas recycling and polarized distribution was not influenced. Our results indicate that proteins with tyrosine- and leucine-based sorting signals can be transported along common saturable pathways.     

Prosaposin facilitates sortilin-independent lysosomal trafficking of progranulin

Mutations in the progranulin (PGRN) gene have been linked to two distinct neurodegenerative diseases, frontotemporal lobar degeneration (FTLD) and neuronal ceroid lipofuscinosis (NCL). Accumulating evidence suggests a critical role of PGRN in lysosomes. However, how PGRN is trafficked to lysosomes is still not clear. Here we report a novel pathway for lysosomal delivery of PGRN. We found that prosaposin (PSAP) interacts with PGRN and facilitates its lysosomal targeting in both biosynthetic and endocytic pathways via the cation-independent mannose 6-phosphate receptor and low density lipoprotein receptor-related protein 1. PSAP deficiency in mice leads to severe PGRN trafficking defects and a drastic increase in serum PGRN levels. We further showed that this PSAP pathway is independent of, but complementary to, the previously identified PGRN lysosomal trafficking mediated by sortilin. Collectively, our results provide new understanding on PGRN trafficking and shed light on the molecular mechanisms behind FTLD and NCL caused by PGRN mutations.     

Sphingolipid trafficking and protein sorting in epithelial cells

Sphingolipids represent a minor, but highly dynamic subclass of lipids in all eukaryotic cells. They are involved in functions that range from structural protection to signal transduction and protein sorting, and participate in lipid raft assembly. In polarized epithelial cells, which display an asymmetric apical and basolateral membrane surface, rafts have been proposed as a sorting principle for apical resident proteins, following their biosynthesis. However, raft-mediated trafficking is ubiquitous in cells. Also, sphingolipids per se, which are strongly enriched in the apical domain, are subject to sorting in polarity development. Next to the trans Golgi network, a subapical compartment called SAC or common endosome appears instrumental in regulating these sorting events.     

Myelin biogenesis: sorting out protein trafficking

Myelin biogenesis is a complex process involving coordinated exocytosis, endocytosis, mRNA transport and cytoskeletal dynamics. Recent studies indicate that soluble neuronal signals may control the surface expression of proteolipid protein, a process that involves reduced endocytosis and/or increased transport carrier recruitment from an intracellular pool.     

Genetic analysis of lysosomal trafficking in Caenorhabditis elegans

The intestinal cells of Caenorhabditis elegans embryos contain prominent, birefringent gut granules that we show are lysosome-related organelles. Gut granules are labeled by lysosomal markers, and their formation is disrupted in embryos depleted of AP-3 subunits, VPS-16, and VPS-41. We define a class of gut granule loss (glo) mutants that are defective in gut granule biogenesis. We show that the glo-1 gene encodes a predicted Rab GTPase that localizes to lysosome-related gut granules in the intestine and that glo-4 encodes a possible GLO-1 guanine nucleotide exchange factor. These and other glo genes are homologous to genes implicated in the biogenesis of specialized, lysosome-related organelles such as melanosomes in mammals and pigment granules in Drosophila. The glo mutants thus provide a simple model system for the analysis of lysosome-related organelle biogenesis in animal cells.     

Targeting of lysosomal proteins

Proper cell homeostasis requires the efficient transport of a large variety of soluble acid hydrolases and transmembrane proteins from the trans-Golgi network (TGN) to lysosomes. While most of these molecules reach this degradative compartment, some transmembrane proteins, in particular, the acid hydrolase receptors are retrieved to the TGN. This bidirectional transport process involves the formation of several vesicular transport intermediates in which cargo molecules are selectively packaged. This review summarizes our current understanding of the molecular mechanisms leading to the proper targeting of lysosomal proteins.     

Differential beta-arrestin trafficking and endosomal sorting of somatostatin receptor subtypes

The physiological responses of somatostatin are mediated by five different G protein-coupled receptors. Although agonist-induced endocytosis of the various somatostatin receptor subtypes (sst(1)-sst(5)) has been studied in detail, little is known about their postendocytic trafficking. Here we show that somatostatin receptors profoundly differ in patterns of beta-arrestin mobilization and endosomal sorting. The beta-arrestin-dependent trafficking of the sst(2A) somatostatin receptor resembled that of a class B receptor in that upon receptor activation, beta-arrestin and the receptor formed stable complexes and internalized together into the same endocytic vesicles. This pattern was dependent on GRK2 (G protein-coupled receptor kinase 2)-mediated phosphorylation of a cluster of phosphate acceptor sites within the cytoplasmic tail of the sst(2A) receptor. Unlike other class B receptors, however, the sst(2A) receptor was rapidly resensitized and recycled to the plasma membrane. The beta-arrestin mobilization of the sst(3) and the sst(5) somatostatin receptors resembled that of a class A receptor in that upon receptor activation, beta-arrestin and the receptor formed relatively unstable complexes that dissociated at or near the plasma membrane. Consequently, beta-arrestin was excluded from sst(3)-containing vesicles. Unlike other class A receptors, a large proportion of sst(3) receptors was subject to ubiquitin-dependent lysosomal degradation and did not rapidly recycle to the plasma membrane. The sst(4) somatostatin receptor is unique in that it did not exhibit agonist-dependent receptor phosphorylation and beta-arrestin recruitment. Together, these findings may provide important clues about the regulation of receptor responsiveness during long-term administration of somatostatin analogs.     

Multicolour imaging of post-Golgi sorting and trafficking in live cells

The biogenesis and maintenance of asymmetry is crucial to many cellular functions including absorption and secretion, signalling, development and morphogenesis. Here we have directly visualized the segregation and trafficking of apical (glycosyl phosphatidyl inositol-anchored) and basolateral (vesicular stomatitis virus glycoprotein) cargo in living cells using multicolour imaging of green fluorescent protein variants. Apical and basolateral cargo segregate progressively into large domains in Golgi/trans-Golgi network structures, exclude resident proteins, and exit in separate transport containers. These remain distinct and do not merge with endocytic structures suggesting that lateral segregation in the trans-Golgi network is the primary sorting event. Fusion with the plasma membrane was detected by total internal reflection microscopy and reveals differences between apical and basolateral carriers as well as new 'hot spots' for exocytosis.     

Mechanisms of procollagen and HSP47 sorting during ER-to-Golgi trafficking

Efficient quality control and export of procollagen from the cell is crucial for extracellular matrix homeostasis, yet it is still incompletely understood. One of the debated questions is the role of a collagen-specific ER chaperone HSP47 in these processes. Most ER chaperones preferentially bind to unfolded polypeptide chains, enabling selective export of natively folded proteins from the ER after chaperone release. In contrast, HSP47 preferentially binds to the natively folded procollagen and is believed to be released only in the ER-Golgi intermediate compartment (ERGIC) or cis-Golgi. HSP47 colocalization with procollagen in punctate structures observed by immunofluorescence imaging of fixed cells has thus been interpreted as evidence for HSP47 export from the ER together with procollagen in transport vesicles destined for ERGIC or Golgi. To understand the mechanism of this co-trafficking and its physiological significance, we imaged the dynamics of fluorescently tagged type I procollagen and HSP47 punctate structures in live MC3T3 murine osteoblasts with up to 120 nm spatial and 500 ms time resolution. Contrary to the prevailing model, we discovered that most bona fide carriers delivering procollagen from ER exit sites (ERESs) to Golgi contained no HSP47, unless the RDEL signal for ER retention in HSP47 was deleted or mutated. These transport intermediates exhibited characteristic rapid, directional motion along microtubules, while puncta with colocalized HSP47 and procollagen similar to the ones described before had only limited, stochastic motion. Live cell imaging and fluorescence recovery after photobleaching revealed that the latter puncta (including the ones induced by ARF1 inhibition) were dilated regions of ER lumen, ERESs, or autophagic structures surrounded by lysosomal membranes. Procollagen was colocalized with HSP47 and ERGIC53 at ERESs. It was colocalized with ERGIC53 but not HSP47 in Golgi-bound transport intermediates. Our results suggest that procollagen and HSP47 sorting occurs at ERES before procollagen is exported from the ER in Golgi-bound transport intermediates, providing new insights into mechanisms of procollagen trafficking.     

The novel Drosophila lysosomal enzyme receptor protein mediates lysosomal sorting in mammalian cells and binds mammalian and Drosophila GGA adaptors

Biogenesis of lysosomes depends in mammalian cells on the specific recognition and targeting of mannose 6-phosphate-containing lysosomal enzymes by two mannose 6-phosphate receptors (MPR46, MPR300), key components of the extensively studied receptor-mediated lysosomal sorting system in complex metazoans. In contrast, the biogenesis of lysosomes is poorly investigated in the less complex metazoan Drosophila melanogaster. We identified the novel type I transmembrane protein lysosomal enzyme receptor protein (LERP) with partial homology to the mammalian MPR300 encoded by Drosophila gene CG31072. LERP contains 5 lumenal repeats that share homology to the 15 lumenal repeats found in all identified MPR300. Four of the repeats display the P-lectin type pattern of conserved cysteine residues. However, the arginine residues identified to be essential for mannose 6-phosphate binding are not conserved. The recombinant LERP protein was expressed in mammalian cells and displayed an intracellular localization pattern similar to the mammalian MPR300. The LERP cytoplasmic domain shows highly conserved interactions with Drosophila and mammalian GGA adaptors known to mediate Golgi-endosome traffic of MPRs and other transmembrane cargo. Moreover, LERP rescues missorting of soluble lysosomal enzymes in MPR-deficient cells, giving strong evidence for a function that is equivalent to the mammalian counterpart. However, unlike the mammalian MPRs, LERP did not bind to the multimeric mannose 6-phosphate ligand phosphomannan. Thus ligand recognition by LERP does not depend on mannose 6-phosphate but may depend on a common feature present in mammalian lysosomal enzymes. Our data establish a potential important role for LERP in biogenesis of Drosophila lysosomes and suggest a GGA function also in the receptor-mediated lysosomal transport system in the fruit fly.     

The role of motor proteins in endosomal sorting

Microtubule motor proteins play key roles in the spatial organization of intracellular organelles as well as the transfer of material between them. This is well illustrated both by the vectorial transfer of biosynthetic cargo from the endoplasmic reticulum to the Golgi apparatus as well as the sorting of secretory and endocytic cargo in the endosomal system. Roles have been described for dynein and kinesin motors in each of these steps. Cytoplasmic dynein is a highly complex motor comprising multiple subunits that provide functional specialization. The family of human kinesins includes over 40 members. This complexity provides immense functional diversity, yet little is known of the specific requirements and functions of individual motors during discrete membrane trafficking steps. In the present paper, we describe some of the latest findings in this area that seek to define the mechanisms of recruitment and control of activity of microtubule motors in spatial organization and cargo trafficking through the endosomal network.