Yol082p, a novel CVT protein involved in the selective targeting of aminopeptidase I to the yeast vacuole.

The yeast vacuolar enzyme aminopeptidase I (API) is synthesized in the cytoplasm as a precursor (pAPI). Upon its assembly into dodecamers, pAPI is wrapped by double-membrane saccular structures for its further transport within vesicles that fuse with the vacuolar membrane and release their content in the vacuolar lumen. Targeting of API to the vacuole occurs by two alternative transport routes, the cvt and the autophagy pathways, which although mechanistically similar specifically operate under vegetative growth or nitrogen starvation conditions, respectively. We have studied the role of Yol082p, a protein identified by its ability to interact with API, in the transport of its precursor to the vacuole. We show that Yol082p interacts with mature API, an interaction that is strengthened by the amino extension of the API protein. Yol082p is required for targeting of pAPI to the vacuole, both under growing and short term nitrogen starvation conditions. Absence of Yol082p does not impede the assembly of pAPI into dodecamers, but precludes the enclosure of pAPI within transport vesicles. Microscopy studies show that during vegetative growth Yol082p is distributed between a cytoplasmic pool and a variable number of 0.13--0.27-microm round, mobile structures, which are no longer observed under conditions of nitrogen starvation, and become larger in cells expressing the inactive Yol082 Delta C32p, or lacking Apg12p. In contrast to the autophagy mutants involved in API transport, a Delta yol082 strain does not lose viability under nitrogen starvation conditions, indicating normal function of the autophagy pathway. The data are consistent with a role of Yol082p in an early step of the API transport, after its assembly into dodecamers. Because Yol082p fulfills the functional requisites that define the CVT proteins, we propose to name it Cvt19.

Moving the insulin-regulated glucose transporter GLUT4 into and out of storage.

The glucose transporter isoform GLUT4 is unique among the glucose transporter family of proteins in that, in resting cells, it is sequestered very efficiently in a storage compartment. In insulin-sensitive cells, such as fat and muscle, insulin stimulation leads to release of GLUT4 from this reservoir and its translocation to the plasma membrane. This process is crucial for the control of blood and tissue glucose levels. Investigations of the composition and structure of the GLUT4 storage compartment, together with the targeting motifs that direct GLUT4 to this compartment, have been extensive but have been controversial. Recent findings have now provided a clearer consensus of opinion on the mechanisms involved in the formation of this storage compartment. However, another controversy has now emerged, which is unresolved. This concerns the issue of whether the insulin-regulated step occurs at the level of release of GLUT4 from the storage compartment or at the level at which released vesicles fuse with the plasma membrane.

Targeting motifs in GLUT4 (review).

The trafficking of the insulin-sensitive glucose transporter, GLUT4, is the paradigm of how cells control the movement of membrane proteins through intricate pathways of transport in response to external stimuli, and how, by doing so, regulate their function. The GLUT4 intracellularly sequestered in resting adipocytes and muscle cells becomes exposed on their surface in response to an increase in insulin levels and muscle contraction, where it facilitates glucose uptake. Ceasing of the stimuli is followed by endocytosis of the GLUT4 molecules exposed on the plasma membrane and their recycling to the original stores, where they are retained. This review discusses current understanding of the organelles that host GLUT4 and the motifs that mediate its trafficking.

Recycling of the insulin-sensitive glucose transporter GLUT4. Access of surface internalized GLUT4 molecules to the perinuclear storage compartment is mediated by the Phe5-Gln6-Gln7-Ile8 motif.

The insulin-sensitive glucose transporter GLUT4 is translocated to the plasma membrane in response to insulin and recycled back to the intracellular store(s) after removal of the hormone. We have used clonal 3T3-L1 fibroblasts and adipocyte-like cells stably expressing wild-type GLUT4 to characterize (a) the intracellular compartment where the bulk of GLUT4 is intracellularly stored and (b) the mechanisms involved in the recycling of endocytosed GLUT4 to the store compartment. Surface internalized GLUT4 is targeted to a large, flat, fenestrated saccular structure resistant to brefeldin A that localized to the vicinity of the Golgi complex is sealed to endocytosed transferrin (GLUT4 storage compartment). Recycling of endocytosed GLUT4 was studied by comparing the cellular distributions of antibody/biotin tagged GLUT4 and GLUT4(Ser(5)), a mutant with the Phe(5)-Gln(6)-Gln(7)-Ile(8) inactivated by the substitution of Ser for Phe(5). Ablation of the Phe(5)-Gln(6)-Gln(7)-Ile(8) inhibits the recycling of endocytosed GLUT4 to the GLUT4 store compartment and results in its transport to late endosomes/lysosomes where it is rapidly degraded.

Distinct reading of different structural determinants modulates the dileucine-mediated transport steps of the lysosomal membrane protein LIMPII and the insulin-sensitive glucose transporter GLUT4.

Leucine-based motifs mediate the sorting of membrane proteins at such cellular sites as the trans-Golgi network, endosomes, and plasma membrane. A Leu paired with a second Leu, Ile, or Met, while itself lacking the ability to mediate transport, is the key structural feature in these motifs. Here we have studied the structural differences between the leucine-based motifs contained in the COOH tails of LIMPII and GLUT4, two membrane proteins that are transported through the secretory pathway and are targeted to lysosomes () and to a perinuclear compartment adjacent to the Golgi complex (), respectively. LIMPII and GLUT4 display negatively (Asp(470)/Glu(471)) and positively (Arg(484)/Arg(485)) charged residues, respectively, at positions -4 and -5 upstream from the critical Leu residue. The change in the charge sign of residues -4 and -5 results in missorting of LIMPII and GLUT4. We note that the acidic Glu residue at position -4 is critical for efficient intracellular sorting of LIMPII to lysosomes, but is dispensable for its surface internalization by endocytosis. Efficient intracellular sorting and endocytosis of GLUT4 require an Arg pair between positions -4 and -7. These results are consistent with the existence of distinct leucine-based motifs and provide evidence of their different readings at different cellular sites.

Targeting of aminopeptidase I to the yeast vacuole is mediated by Ssa1p, a cytosolic member of the 70-kDa stress protein family.

The two cytosolic members of the highly conserved 70-kDa stress protein family, Ssa1p and Ssa2p, were specifically retained by the prepro-NH(2) extension of the vacuolar aminopeptidase I precursor (pAPI) conjugated to agarose (Sulfolink). A temperature-sensitive mutant strain a1(ts)a234 (ssa1(ts) ssa2 ssa3 ssa4), when incubated at the restrictive temperature, was able to assemble the API precursor into dodecamers, but failed to pack pAPI into vesicles and to convert it into mature API (mAPI), a process that occurs in the vacuole. Altogether these results indicate that Ssa1p mediates the targeting of pAPI to the vacuole.

Intracellular targeting and retention of the glucose transporter GLUT4 by the perinuclear storage compartment involves distinct carboxyl-tail motifs.

The mechanisms by which the insulin-sensitive glucose transporter, GLUT4, is targeted and retained in a storage compartment near to the Golgi complex are poorly understood. Here we report that removal of the carboxyl-terminal acidic Pro(505)AspGluAsnAsp(509) sequence prevents the storage of GLUT4 in the VAMP-2 positive compartment adjacent to the Golgi complex (GSC), and results in its targeting to GLUT4-positive vesicles and Rab7-positive late endosomes. Storage of the truncated GLUT4 in the GSC is restored by substitution of Phe for the Tyr(502) residue adjacent to Pro(505) or by treatment of cells with the tyrosine kinase inhibitor genistein. Ablation of the Leu(489)Leu(490)-based motif prevents the targeting of GLUT4delta5 to GLUT4-positive-vesicles and late endosomes as well as the retention of GLUT4delta5Phe(502 )by the GSC. These results are consisting with a model of GLUT4 transport in which the targeting of the protein from the TGN to the GSC is mediated by the Leu(489)Leu(490)-based motif and its release from the GSC involves Tyr(502 )and the adjacent carboxyl-terminal Pro(505)AspGluAsnAsp(509) sequence.

Membrane flow through the Golgi apparatus: specific disassembly of the cis-Golgi network by ATP depletion.

Incubation of NRK cells for 30 to 45 minutes with 50 mM 2-deoxy-D-glucose (DOG) in glucose and pyruvate-free medium results in depletion of the cellular ATP pool and in specific disassembly of the cis-Golgi network (CGN), with the stack of Golgi cisternae (SGC) and the trans-Golgi network (TGN) remaining intact and sensitive to BFA. The disassembly of the CGN is mediated by long tubular structures extending outwards from the Golgi complex and involves microtubules. Upon removal of DOG and addition of glucose and pyruvate to the culture medium, the morphology of the CGN is slowly reestablished. Reconstruction of the CGN involves COPI/COPII-positive vesicles that resume the transport of proteins and in particular of CGN membrane proteins out of the ER. Exit of CGN membrane proteins from the ER is insensitive to BFA. In cells pretreated with nocodazole, the CGN membrane proteins are transported to the vicinity of the SGC fragments dispersed throughout the cytoplasm. Ultrastructural studies of cells engaged in the reconstruction of the CGN revealed that the CGN cisterna emerge as tubular structures extending from 0.2-0.3 microm uncoated vesicles prior to their organization on the cis-side of the SGC.

The prepropeptide of vacuolar aminopeptidase I is necessary and sufficient to target the fluorescent reporter protein GFP to the vacuole of yeast by the Ccvt pathway.

We have studied the capacity of the prepro amino extension of vacuolar protease leucine aminopeptidase I (API) to target the fluorescent reporter protein GFP to the vacuole of yeast. The preproGFP chimera constructed by extending the amino end of GFP with the prepro-part of API is rapidly degraded in both wild-type WCG cells and WCG 11/21a cells deficient in the proteasome. In contrast, the chimera expressed in WCG-PP cells deficient in both proteasome activity and vacuolar proteinase A accumulates in the vacuole, where it remains stable. Replacement of Gly by Ile-7, a substitution that prevents folding of the pre-part into an amphipathic helix and inhibits the targeting of the API precursor to the vacuole, inhibits the targeting of preproGFP to the vacuole. The separated pre- and pro-parts of the API precursor do not target GFP to the vacuole. Targeting of preproGFP to the vacuole is independent of its levels of expression, as the fluorescent protein localizes to the vacuole in cells expressing the protein under the control of both the GAL 1/10 or the API promoter. The preproGFP expressed under both promoters is recovered as monomers from cytosolic cell extracts. PreproGFP expressed under the API promoter is packed into cytoplasmic bodies that penetrate into the vacuolar lumen to release the protein. Altogether our results show that the prepro-part of the API precursor is necessary and sufficient to target the green fluorescent reporter protein to the vacuole.

Localization of atypical protein kinase C isoforms into lysosome-targeted endosomes through interaction with p62.

An increasing number of independent studies indicate that the atypical protein kinase C (PKC) isoforms (aPKCs) are critically involved in the control of cell proliferation and survival. The aPKCs are targets of important lipid mediators such as ceramide and the products of the PI 3-kinase. In addition, the aPKCs have been shown to interact with Ras and with two novel proteins, LIP (lambda-interacting protein; a selective activator of lambda/iotaPKC) and the product of par-4 (a gene induced during apoptosis), which is an inhibitor of both lambda/iotaPKC and zetaPKC. LIP and Par-4 interact with the zinc finger domain of the aPKCs where the lipid mediators have been shown to bind. Here we report the identification of p62, a previously described phosphotyrosine-independent p56(lck) SH2-interacting protein, as a molecule that interacts potently with the V1 domain of lambda/iotaPKC and, albeit with lower affinity, with zetaPKC. We also show in this study that ectopically expressed p62 colocalizes perfectly with both lambda/iotaPKC and zetaPKC. Interestingly, the endogenous p62, like the ectopically expressed protein, displays a punctate vesicular pattern and clearly colocalizes with endogenous lambda/iotaPKC and endogenous zetaPKC. P62 colocalizes with Rab7 and partially with lamp-1 and limp-II as well as with the epidermal growth factor (EGF) receptor in activated cells, but not with Rab5 or the transferrin receptor. Of functional relevance, expression of dominant negative lambda/iotaPKC, but not of the wild-type enzyme, severely impairs the endocytic membrane transport of the EGF receptor with no effect on the transferrin receptor. These findings strongly suggest that the aPKCs are anchored by p62 in the lysosome-targeted endosomal compartment, which seems critical for the control of the growth factor receptor trafficking. This is particularly relevant in light of the role played by the aPKCs in mitogenic cell signaling events.

Demonstration of a Ca2+ requirement for thyroglobulin dimerization and export to the golgi complex.

We have examined the effects of depleting the endoplasmic reticulum Ca2+ store on the maturation of newly synthesized thyroglobulin molecules, their export to the Golgi complex, and their secretion by FRTL-5 cells. An inhibitor of the endoplasmic reticulum Ca2+ pump, thapsigargin, and the Ca2+ ionophore A23187 depleted the endoplasmic reticulum Ca2+ store and strongly inhibited thyroglobulin secretion in cells chased in medium containing 0.1 mM Ca2+. Inhibition of thyroglobulin secretion was caused by a block in the export of newly synthesized thyroglobulin molecules from the endoplasmic reticulum to the Golgi complex, as shown by cell-fractionation experiments and the intracellular accumulation of endoH-sensitive thyroglobulin. The thyroglobulin molecules retained in the endoplasmic reticulum of cells treated with the drugs were found to assemble more slowly into dimers than thyroglobulin in control cells. Protease-sensitivity experiments demonstrated that thyroglobulin dimers assembled in the presence of thapsigargin had a different conformation with respect to dimers assembled in controls cells.

A di-leucine-based motif in the cytoplasmic tail of LIMP-II and tyrosinase mediates selective binding of AP-3.

Among the various coats involved in vesicular transport, the clathrin associated coats that contain the adaptor complexes AP-1 and AP-2 are the most extensively characterized. The function of the recently described adaptor complex AP-3, which is similar to AP-1 and AP-2 in protein composition but does not associate with clathrin, is not known. By monitoring surface plasmon resonance we observed that AP-3 is able to interact with the tail of the lysosomal integral membrane protein LIMP-II and that this binding depends on a DEXXXLI sequence in the LIMP-II tail. Furthermore, AP-3 bound to the cytoplasmic tail of the melanosome-associated protein tyrosinase which contains a related EEXXXLL sequence. The tails of LIMP-II and tyrosinase either did not interact, or only interacted poorly, with AP-1 or AP-2. In contrast, the cytoplasmic tails of other membrane proteins containing di-leucine and/or tyrosine-based sorting signals did not bind AP-3, but AP-1 and/or AP-2. This points to a function of AP-3 in intracellular sorting to lysosomes and melanosomes of a subset of cargo proteins via di-leucine-based sorting motifs.

Poliovirus infection and expression of the poliovirus protein 2B provoke the disassembly of the Golgi complex, the organelle target for the antipoliovirus drug Ro-090179.

Infection of Vero cells with poliovirus results in complete disassembly of the Golgi complex. Milestones of the process of disassembly are the release to the cytosol of the beta-COP bound to Golgi membranes, the disruption of the cis-Golgi network into fragments scattered throughout the cytoplasm, and the disassembly of the stacked cisternae by a process mediated by long tubular structures. Transient expression of the viral protein 2B in COS-7 cells also causes the disassembly of the Golgi complex by a process preceded by the accumulation of the protein in the Golgi area. Vero cells infected for 3 h show no recognizable Golgi complexes at the ultrastructural level and display an enormously swollen endoplasmic reticulum (ER) with extensive areas of its surface heavily coated. Ro-090179 (Ro), a flavonoid isolated from the herb Agastache rugosa, provokes the specific swelling and disruption of the Golgi complex and strongly inhibits poliovirus infection. Ro provokes the swelling and the disruption of the stacked cisternae and trans-Golgi elements without affecting the cis-most Golgi cisternae much. Moreover, Ro inhibits the fusion of the Golgi complex with the ER in cells treated with brefeldin A and provokes the accumulation of the intermediate compartment membrane protein p58 into ERD2-positive Golgi elements but has no effect on the anterograde transport involved in protein secretion. Our results indicate that the secretory pathway and specifically the Golgi complex are preferential targets of poliovirus.

Folding of the presequence of yeast pAPI into an amphipathic helix determines transport of the protein from the cytosol to the vacuole.

To investigate the role of the 17 residues long presequence (p17) in the transport of the precursor of yeast API (pAPI) from the cytosol to the vacuole we have studied the effects of point mutations upon its conformation and on the process of transport. 1H NMR analysis of p17 indicates that in aqueous solution 26% of the molecules have the 4-12 segment folded into an helix. The hydrophobic environment provided by SDS micelles promotes the folding of 54% of the p17 molecules into a 5-16 amphipathic alpha-helix. Both Schiffer-Edmunson helical wheel analysis of segment 4-12 and residue hydrophobic moments calculated considering all possible side-chain orientations between 80 and 120 degrees, indicate the amphipathic character of the helixes assembled in water and detergent. Charge interactions between the dipole pairs N-Glu2Glu3 and C-Lys12Lys13 are essential for helix stability and condition pAPI transport. Substitution of either Pro2Pro3 or Lys2Lys3 for Glu2Glu3, results in moderate destabilization of the helix, decreases protein targeting to the vacuolar membrane and partly inhibits translocation of the protein to the vacuolar lumen. Replacement of either Pro12Pro13 or Glu12Glu13 for Lys12Lys13, causes a major disruption of the helix, decreases protein targeting and blocks completely the translocation of the protein to the vacuolar lumen. Replacement of Gly7 for Ile7, a substitution which is known to destabilize alpha-helixes in peptides and proteins as a result of the peptide bond to the solvent at Gly residues, produces similar effects as the substitutions for the K12K13 pair. The effects of Gly7 on helix stability and protein transport are partly reversed by introduction of Asp residues at positions 2 and 3 and Ala at position 4. Replacements such as Arg2 for Glu2, or Arg6 for Glu6, which change the net and local charges of the presequence without altering its conformation, have no effect on the protein transport. These results provide direct evidence of the involvement of the presequence in the transport of pAPI from the cytosol to the vacuole. They show that folding of the pAPI presequence is conditioned by the physical/chemical properties of the environment and is critical for targeting the protein to the vacuolar membrane and for its translocation to the vacuolar lumen.

Yeast aminopeptidase I is post-translationally sorted from the cytosol to the vacuole by a mechanism mediated by its bipartite N-terminal extension.

Transport of aminopeptidase I (API) to the vacuole appears to be insensitive to blockage of the secretory pathway. Here we show that the N-terminal extension of the 61 kDa precursor of API (pAPI) is proteolytically processed in two sequential steps. The first step involves proteinase A (PrA) and produces a 55 kDa unstable intermediate (iAPI). The second step involves proteinase B (PrB) and converts iAPI into the 50 kDa stable, mature enzyme (mAPI). Reversion of the cup1 growth phenotype by a pAPI-CUP1 chimera indicates that pAPI is transported to the vacuole by a post-translational mechanism. Deletion of the first 16 amino acids results in accumulation of the truncated protein in the cytosol, indicating that pAPI is actively transported to the vacuole. The chimera pAPI-myc, constructed by fusing a myc tag to the C-terminus of pAPI, was exploited to dissect the mechanism of pAPI transport. Cell fractionation studies show the presence of iAPI-myc and mAPI in a fraction of vacuoles purified by density centrifugation. This and the sequential conversion of pAPI-myc into iAPI-myc and mAPI lacking the myc tag is consistent with insertion of pAPI into the vacuolar membrane through its N-terminal extension. The specific mechanism of API sorting demonstrates a new pathway of protein transport in vacuolar biogenesis.

Targeting of membrane proteins to endosomes and lysosomes.

The pathways involved in targeting membrane proteins to lysosomes are extraordinarily complex. Newly synthesized proteins in the ER are transported to the Golgi complex, and upon arrival at the trans Golgi network (TGN) are targeted either directly to endosomes, or first to the cell surface from where they can be rapidly internalized into the endocytic pathway for delivery to lysosomes. The routes to endosomes are specified by sorting motifs in the cytoplasmic tails of the proteins that are recognized at the TGN or plasma membrane. The molecular details of these processes are just emerging.

The residues Leu(Ile)475-Ile(Leu, Val, Ala)476, contained in the extended carboxyl cytoplasmic tail, are critical for targeting of the resident lysosomal membrane protein LIMP II to lysosomes.

LIMP II, a type II lysosomal integral membrane protein, and the CD36/LIMP II construct are targeted to lysosomes by means of a signal expressed in the tyrosine-lacking carboxyl cytoplasmic tail of LIMP II (Vega, M. A., Rodriguez, F., Seguí, B., Calés, C., Alcalde, J., and Sandoval, I. V. (1991) J. Biol. Chem. 266, 16269-16272; Vega, M. A., Seguí-Real, B., Garcia, J. A., Calés, C., Rodriguez, F., Vandekerckhove, J., and Sandoval, I. V. (1991) J. Biol. Chem. 266, 16818-16824). Substitution of Leu475 with Ile resulted in a decreased efficiency of targeting. Mutant forms produced by substituting Leu475 by hydrophobic residues with either large (Val) or small (Ala, Gly) side chains, or by a charged residue (Asp), showed inhibited targeting. In contrast, the contiguous Ile476 residue could be replaced by either Leu, without loss in the efficiency of targeting, or by Val or Ala, with some impediment. Substitution of Ile476 by either Gly or Asp inhibited completely the targeting. The addition of the sequence Ser-Trp-Asp to the carboxyl end of the construct did not interfere with targeting. Data from 1H NMR analysis of the icosapeptide corresponding to the carboxyl cytoplasmic tail of LIMP II indicated the predominance of structures with extended random coil conformations, suggesting that the targeting signal is contained in a domain with an extended configuration.

gp74 a membrane glycoprotein of the cis-Golgi network that cycles through the endoplasmic reticulum and intermediate compartment.

A monoclonal antibody CC92 (IgM), raised against a fraction of rat liver enriched in Golgi membranes, recognizes a novel Endo H-resistant 74-kD membrane glycoprotein (gp74). The bulk of gp74 is confined to the cis-Golgi network (CGN). Outside the Golgi gp74 is found in tubulovesicular structures and ER foci. In cells incubated at 37 degrees C the majority of gp74 is segregated from the intermediate compartment (IC) marker p58. However, in cells treated with organelle perturbants such as low temperature, BFA, and [AIF4]- the patterns of the two proteins become indistinguishable. Both proteins are retained in the Golgi complex at 20 degrees C and in the IC at 15 degrees C. Incubation of cells with BFA results in relocation of gp74 to p58 positive IC elements. [AIF4]- induces the redistribution of gp74 from the Golgi to p58-positive vesicles and does not retard the translocation of gp74 to IC elements in cells treated with BFA. Disruption of microtubules by nocodazol results in the rapid disappearance of the Golgi elements stained by gp74 and redistribution of the protein into vesicle-like structures. The responses of gp74 to cell perturbants are in sharp contrast with those of cis/middle and trans-Golgi resident proteins whose location is not affected by low temperatures or [AIF4]-, are translocated to the ER upon addition of BFA, and stay in slow disintegrating Golgi elements in cells treated with nocodazol. The results suggest that gp74 is an itinerant protein that resides most of the time in the CGN and cycles through the ER/IC following the pathway used by p58.

Assembly and disassembly of the Golgi complex: two processes arranged in a cis-trans direction.

We have studied the disassembly and assembly of two morphologically and functionally distinct parts of the Golgi complex, the cis/middle and trans cisterna/trans network compartments. For this purpose we have followed the redistribution of three cis/middle- (GMPc-1, GMPc-2, MG 160) and two trans- (GMPt-1 and GMPt-2) Golgi membrane proteins during and after treatment of normal rat kidney (NRK) cells with brefeldin A (BFA). BFA induced complete disassembly of the cis/middle- and trans-Golgi complex and translocation of GMPc and GMPt to the ER. Cells treated for short times (3 min) with BFA showed extensive disorganization of both cis/middle- and trans-Golgi complexes. However, complete disorganization of the trans part required much longer incubations with the drug. Upon removal of BFA the Golgi complex was reassembled by a process consisting of three steps: (a) exist of cis/middle proteins from the ER and their accumulation into vesicular structures scattered throughout the cytoplasm; (b) gradual relocation and accumulation of the trans proteins in the vesicles containing the cis/middle proteins; and (c) assembly of the cisternae, and reconstruction of the Golgi complex within an area located in the vicinity of the centrosome from which the ER was excluded. Reconstruction of the cis/middle-Golgi complex occurred under temperature conditions inhibitory of the reorganization of the trans-Golgi complex, and was dependent on microtubules. Reconstruction of the trans-Golgi complex, disrupted with nocodazole after selective fusion of the cis/middle-Golgi complex with the ER, occurred after the release of cis/middle-Golgi proteins from the ER and the assembly of the cis/middle cisternae.

Targeting of lysosomal integral membrane protein LIMP II. The tyrosine-lacking carboxyl cytoplasmic tail of LIMP II is sufficient for direct targeting to lysosomes.

Time course experiments of the localization of rat LIMP II expressed in COS cells show that the protein is transported directly from the Golgi complex to lysosomes. Substitution of the tyrosine-lacking carboxyl cytoplasmic tail of LIMP II for the native cytoplasmic tails of the plasma membrane proteins CD36 and CD8 resulted in straight transport of both proteins to lysosomes. The synthetic tyrosine-containing heptapeptide, RGTGVYG, did not replace the natural carboxyl cytoplasmic tail of LIMP II in its ability to transport both CD36 and CD8 to lysosomes, and the two constructs were transported to and expressed at the plasma membrane. Substitution of the cytoplasmic tails of either CD36 or CD8 for the carboxyl cytoplasmic tail of LIMP II resulted in transport of the mutants to the plasma membrane where they underwent endocytosis before accumulating into lysosomes. The results indicate that a motif contained in the tyrosine-lacking carboxyl cytoplasmic tail of LIMP II is sufficient to target proteins directly from the Golgi complex to lysosomes.