AP-3 adaptor functions in targeting P-selectin to secretory granules in endothelial cells

P-selectin, a cell adhesion protein participating in the early stages of inflammation, contains multiple sorting signals that regulate its cell surface expression. Targeting to secretory granules regulates delivery of P-selectin to the cell surface. Internalization followed by sorting from early to late endosomes mediates rapid removal of P-selectin from the surface. We show here that the P-selectin cytoplasmic domain bound AP-2 and AP-3 adaptor complexes in vitro . The amino acid substitution L768A, which abolishes endosomal sorting and impairs granule targeting of P-selectin, reduced binding of AP-3 adaptors but not AP-2 adaptors. Turnover of P-selectin was 2.4-fold faster than turnover of transferrin receptor in AP-3-deficient mocha fibroblasts, similar to turnover of these two proteins in AP-3-competent cells, demonstrating that AP-3 function is not required for endosomal sorting. However, sorting P-selectin to secretory granules was defective in endothelial cells from AP-3-deficient pearl mice, demonstrating a role for AP-3 adaptors in granule assembly in endothelial cells. P-selectin sorting to platelet α-granules was normal in pearl mice, consistent with earlier evidence that granule targeting of P-selectin is mechanistically distinct in endothelial cells and platelets. These observations establish that AP-3 adaptor functions in assembly of conventional secretory granules, in addition to lysosomes and the 'lysosome-like' secretory granules of platelets and melanocytes.     

A third specificity-determining site in mu 2 adaptin for sequences upstream of Yxx phi sorting motifs

Internalization signals of the YxxΦ type (Φ=bulky hydrophobic side chain) interact with the μ2 chain of AP-2 adaptors. Internalization activity is intolerant of non-conservative substitution of either the tyrosine or the Φ side chains, which bind to hydrophobic pockets in μ2 adaptin in a conformation described as 'a two pinned plug into a socket'. P-selectin, a type I transmembrane protein, contains the YxxΦ-like sequence YGVF in its cytoplasmic domain, but substitution of either the tyrosine or phenylalanine with alanine in the full-length protein causes only small changes in the rate of endocytosis. It is shown here that the sequence YGVF contained within a peptide corresponding to the 17 COOH-terminal amino acids of P-selectin binds to μ2 adaptin in the same fashion previously seen for other YxxΦ motifs. In addition, the P-selectin peptide binds to a third hydrophobic pocket in μ2 adaptin through a leucine at position Y−3 in the peptide. This structure suggests that some sequences can function as a 'three pinned plug', in which internalization activity is not critically dependent on any one of the three interacting side chains.     

Sorting to synaptic-like microvesicles from early and late endosomes requires overlapping but not identical targeting signals

In PC12 neuroendocrine cells, synaptic-like microvesicles (SLMV) are thought to be formed by two pathways. One pathway sorts the proteins to SLMV directly from the plasma membrane (or a specialized domain thereof) in an adaptor protein complex 2-dependent, brefeldin A (BFA)-insensitive manner. Another pathway operates via an endosomal intermediate, involves adaptor protein complex 3, and is BFA sensitive. We have previously shown that when expressed in PC12 cells, HRP-P-selectin chimeras are directed to SLMV mostly via the endosomal, BFA-sensitive route. We have now found that two endosomal intermediates are involved in targeting of HRP-P-selectin chimeras to SLMV. The first intermediate is the early, transferrin-positive, epidermal growth factor-positive endosome, from which exit to SLMV is controlled by the targeting determinants YGVF and KCPL, located within the cytoplasmic domain of P-selectin. The second intermediate is the late, transferrin-negative, epidermal growth factor-positive late endosome, from where HRP-P-selectin chimeras are sorted to SLMV in a YGVF- and DPSP-dependent manner. Both sorting steps, early endosomes to SLMV and late endosomes to SLMV, are affected by BFA. In addition, analysis of double mutants with alanine substitutions of KCPL and YGVF or KCPL and DPSP indicated that chimeras pass sequentially through these intermediates en route both to lysosomes and to SLMV. We conclude that a third site of formation for SLMV, the late endosomes, exists in PC12 cells.     

A di-aromatic motif in the cytosolic tail of the mannose receptor mediates endosomal sorting

The mannose receptor (MR), the prototype of a new family of multilectin receptor proteins important in innate immunity, undergoes rapid internalization and recycling from the endosomal system back to the cell surface. Sorting of the MR in endosomes prevents the receptor from entering lysosomes where it would be degraded. Here, we focused on a diaromatic sequence (Tyr(18)-Phe(19)) in the MR cytoplasmic tail as an endosomal sorting signal. The subcellular distribution of chimeric constructs between the MR and the cation-dependent mannose 6-phosphate receptor was assessed by Percoll density gradients and cell surface assays. Unlike the wild type constructs, mutant receptors with alanine substitutions of Tyr(18)-Phe(19) were highly missorted to lysosomes, indicating that the di-aromatic motif of the MR cytoplasmic tail mediates sorting in endosomes. Within this sequence Tyr(18) is the key residue with Phe(19) contributing to this function. Moreover, Tyr(18) was also found to be essential for internalization, consistent with the presence of overlapping signals for internalization and endosomal sorting in the cytosolic tail of the MR. A di-aromatic amino acid sequence in the cytosolic tail has now been shown to function in two receptors known to be internalized from the plasma membrane, the MR and the cation-dependent mannose 6-phosphate receptor. This feature therefore appears to be a general determinant for endosomal sorting.     

Intracellular sorting and targeting of melanosomal membrane proteins: identification of signals for sorting of the human brown locus protein, gp75

The structural and functional integrity of cytoplasmic organelles is maintained by intracellular mechanisms that sort and target newly synthesized proteins to their appropriate cellular locations. In melanocytic cells, melanin pigment is synthesized in specialized organelles, melanosomes. A family of melanocyte-specific proteins, known as tyrosinase-related proteins that regulate melanin pigment synthesis, is localized to the melanosomal membrane. The human brown locus protein, tyrosinase-related protein-1 or gp75, is the most abundant glycoprotein in melanocytic cells, and is a prototype for melanosomal membrane proteins. To investigate the signals that allow intracellular retention and sorting of glycoprotein (gp)75, we constructed protein chimeras containing the amino-terminal extracellular domain of the T lymphocyte surface protein CD8, and transmembrane and cytoplasmic domains of gp75. In fibroblast transfectants, chimeric CD8 molecules containing the 36-amino acid cytoplasmic domain of gp75 were retained in cytoplasmic organelles. Signals in the gp75 cytoplasmic tail alone, were sufficient for intracellular retention and targeting of the chimeric proteins to the endosomal/lysosomal compartment. Analysis of subcellular localization of carboxy-terminal deletion mutants of gp75 and the CD8/gp75 chimeras showed that deletion of up amino acids from the gp75 carboxyl terminus did not affect intracellular retention and sorting, whereas both gp75 and CD8/gp75 mutants lacking the carboxyl-terminal 27 amino acids were transported to the cell surface. This region contains the amino acid sequence, asn-gln-pro-leu-leu-thr, and this hexapeptide is conserved among other melanosomal proteins. Further evidence showed that this hexapeptide sequence is necessary for intracellular sorting of gp75 in melanocytic cells, and suggested that a signal for sorting melanosomal proteins along the endosomal/lysosomal pathway lies within this sequence. These data provide evidence for common signals for intracellular sorting of melanosomal and lysosomal proteins, and support the notion that lysosomes and melanosomes share a common endosomal pathway of biogenesis.     

A single amino acid substitution in a WW-like domain of diverse members of the PDGF receptor subfamily of tyrosine kinases causes constitutive receptor activation.

Platelet-derived growth factor beta receptor (PDGFbetaR) is a transmembrane receptor tyrosine kinase involved in a variety of cellular functions. We have generated a constitutively activated murine PDGFbetaR containing a valine to alanine substitution at residue 536, located in the cytoplasmic juxtamembrane domain. When this mutant receptor (PR-V536A) was expressed in Ba/F3 cells, it allowed the cells to survive and proliferate in the absence of IL-3 or PDGF, and tyrosine phosphorylation of PR-V536A was increased markedly compared with that of the wild-type PDGFbetaR in the absence of ligand and similar to that observed in ligand-activated PDGFbetaR. PR-V536A displayed increased tyrosine kinase activity in vitro toward an exogenous substrate, and the tyrosine kinase activity of the receptor was required for the constitutive activation of the mutant. This valine to alanine substitution also activated a PDGFbetaR mutant unable to bind PDGF. Alanine substitutions at positions homologous to V536 of the murine PDGFbetaR also activated other members of the PDGF receptor subfamily. The amino acid sequence of this region revealed a strong similarity to WW domains present in other signal transduction proteins. Furthermore, GST fusion proteins containing the juxtamembrane region of the PDGFR specifically associated with peptides containing the WW domain consensus recognition sequence PPXY. The results suggest that the cytoplasmic juxtamembrane domain plays a role in the regulation of receptor activity and function, perhaps by participating in protein-protein interactions.     

Endosomal Sorting of Amyloid Precursor Protein-P-Selectin Chimeras Influences Secretase Processing

Amyloid β protein, the major component of the senile plaques in Alzheimer's disease, is generated by secretory and endocytic processing of amyloid precursor protein. Internalized amyloid precursor protein either recycles to the plasma membrane, where α-secretase resides, or moves to acidic compartment(s) for β-secretase exposure. While the trans-Golgi network contains β-secretase activity, recent examination of the subcellular distribution of this proteinase, called BACE, has led to the suggestion that β-secretase activity might also reside at the plasma membrane and in endosomes. To examine the role of endocytic compartments in β-secretase processing of amyloid precursor protein, the wild-type and endosomal sorting mutant P-selectin cytoplasmic domains were used to control movement of amyloid precursor protein through endosomes. Amyloid precursor protein/P-selectin, which is sorted from early to late endosomes, undergoes significantly less α-secretase cleavage, and more β-secretase cleavage, than amyloid precursor protein/P-selectin768A, a mutant that recycles more efficiently to the cell surface. Our results demonstrate that endosomal sorting influences relative exposure of the amyloid precursor protein/P-selectin chimeras to α- and β-secretase activities, and suggest that, because delivery to late endocytic compartments favors β-secretase processing of amyloid precursor protein, there is likely limited β-secretase activity in early endosomes or at the cell surface. We propose that the trans-Golgi network may be involved in both secretory and endocytic generation of amyloid β protein.     

The cytoplasmic domain of P-selectin contains a sorting determinant that mediates rapid degradation in lysosomes

P-selectin is a cell adhesion molecule required transiently on the surface of activated platelets and endothelial cells as a receptor for leukocytes. It is stored in secretory granules in platelets, endothelial cells, and transfected neuroendocrine cells and is rapidly delivered to the plasma membrane upon exocytosis of the secretory granules. It is then rapidly internalized in endothelial cells and transfected cells. We find that in transfected neuroendocrine PC12 cells, the fraction of P-selectin that is not targeted to secretory granules is rapidly degraded. In transfected CHO fibroblasts, which lack secretory granules, P-selectin was degraded with a half time of 2.3 h in plated cells, while low density lipoprotein receptor (LDL-R) had a half life of 9 h. In cells cultured in ammonium chloride to inhibit lysosomal proteinases, P-selectin was protected from degradation and rapidly accumulated in vesicles enriched in lgp-B, a resident lysosomal membrane protein. The cytoplasmic domain of P- selectin was sufficient to confer rapid turnover on LDL-R. Deletion of 10 amino acids from the cytoplasmic domain of P-selectin extended the half life to 9.5 h and abrogated rapid lysosomal targeting in the presence of ammonium chloride, implicating this sequence as a necessary element of a novel lysosomal targeting signal. The rate limiting step in degradation occurred after internalization from the cell surface, indicating that sorting of P-selectin away from efficiently recycled proteins occurs in endosomes. We propose that this sorting event represents a constitutive equivalent of receptor down regulation, and may function to regulate the expression of P-selectin at the surface of activated endothelial cells.     

Rapid transport of internalized P-selectin to late endosomes and the TGN: roles in regulating cell surface expression and recycling to secretory granules

Prior studies on receptor recycling through late endosomes and the TGN have suggested that such traffic may be largely limited to specialized proteins that reside in these organelles. We present evidence that efficient recycling along this pathway is functionally important for nonresident proteins. P-selectin, a transmembrane cell adhesion protein involved in inflammation, is sorted from recycling cell surface receptors (e.g., low density lipoprotein [LDL] receptor) in endosomes, and is transported from the cell surface to the TGN with a half-time of 20-25 min, six to seven times faster than LDL receptor. Native P-selectin colocalizes with LDL, which is efficiently transported to lysosomes, for 20 min after internalization, but a deletion mutant deficient in endosomal sorting activity rapidly separates from the LDL pathway. Thus, P-selectin is sorted from LDL receptor in early endosomes, driving P-selectin rapidly into late endosomes. P-selectin then recycles to the TGN as efficiently as other receptors. Thus, the primary effect of early endosomal sorting of P-selectin is its rapid delivery to the TGN, with rapid turnover in lysosomes a secondary effect of frequent passage through late endosomes. This endosomal sorting event provides a mechanism for efficiently recycling secretory granule membrane proteins and, more generally, for downregulating cell surface receptors.     

A hydrophobic amino acid cluster inserted into the C-terminus of a recycling cell surface receptor functions as an endosomal sorting signal

Cell surface receptors ubiquitylated after ligand stimulation are internalized and delivered to the lysosomal pathway for degradation. Ubiquitylated receptors are captured by ESCRT protein complexes that sort them to the lysosomal pathway. Hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) is a component of endosomal sorting complexes required for transport (ESCRT)-0 that recognizes ubiquitin attached to receptors, indicating that it functions as a key molecule for ubiquitin-dependent endosomal sorting. In a previous study on interleukin (IL)-2 receptor β (IL-2Rβ) and IL-4 receptor α (IL-4Rα), which are constitutively internalized without ligand stimulation, we revealed that Hrs bound to IL-2Rβ and IL-4Rα in a ubiquitin-independent manner, and identified a hydrophobic amino acid cluster in the cytoplasmic region of IL-2Rβ and IL-4Rα as the Hrs-interacting domain. However, a chimeric receptor containing the hydrophobic amino acid cluster inserted into the C-terminal of IL-2Rα was not delivered to late endosomes, but recycled back to the plasma membrane. In the present study, we explored the functional domain related to endosomal sorting in IL-2Rβ together with the hydrophobic amino acid cluster, and discovered the importance of an approximately 30-amino acid stretch following the C-terminus of the hydrophobic amino acid cluster in IL-2Rβ. Even though the amino acid stretch following the hydrophobic amino acid cluster was composed of arbitrary amino acids, such a stretch was also permissive for the sorting ability, suggesting that the hydrophobic amino acid cluster functions as an endosomal sorting signal. These findings clarify part of the molecular mechanism underlying the ubiquitin-independent endosomal sorting of cytokine receptors that are constitutively internalized without ligand stimulation.     

A new member of the sorting nexin family interacts with the C-terminus of P-selectin

P-selectin is a cell adhesion molecule found in platelets and endothelial cells mediating binding of leukocytes. It is stored in secretory granules and expressed at the plasma membrane after cell activation. After rapid internalisation P-selectin recycles or is degraded. The 35 amino acid cytoplasmic domain of P-selectin contains signals for sorting into secretory granules, for endocytosis and for delivery to lysosomes. To investigate protein-protein interactions, we performed two-hybrid screening using the cytoplasmic domain of P-selectin as bait. KIAA0064 was identified as a putative intracellular P-selectin binding protein. Because the protein contains a phox homology (PX) domain in the N-terminus which is a characteristic feature of the sorting nexin (SNX) family, it was named SNX17. The PX domain is not required for binding of P-selectin in the two-hybrid system. Expression of a fusion protein between SNX17 and green fluorescent protein demonstrated localisation of SNX17 in the cytosol and to membranes.     

Structural Determinants for Binding of Sorting Nexin 17 (SNX17) to the Cytoplasmic Adaptor Protein Krev Interaction Trapped 1 (KRIT1)

Sorting nexin 17 (SNX17) is a member of the family of cytoplasmic sorting nexin adaptor proteins that regulate endosomal trafficking of cell surface proteins. SNX17 localizes to early endosomes where it directly binds NPX(Y/F) motifs in the cytoplasmic tails of its target receptors to mediate their rates of endocytic internalization, recycling, and/or degradation. SNX17 has also been implicated in mediating cell signaling and can interact with cytoplasmic proteins. KRIT1 (Krev interaction trapped 1), a cytoplasmic adaptor protein associated with cerebral cavernous malformations, has previously been shown to interact with SNX17. Here, we demonstrate that SNX17 indeed binds directly to KRIT1 and map the binding to the second Asn-Pro-Xaa-Tyr/Phe (NPX(Y/F)) motif in KRIT1. We further characterize the interaction as being mediated by the FERM domain of SNX17. We present the co-crystal structure of SNX17-FERM with the KRIT1-NPXF2 peptide to 3.0 Å resolution and demonstrate that the interaction is highly similar in structure and binding affinity to that between SNX17 and P-selectin. We verify the molecular details of the interaction by site-directed mutagenesis and pulldown assay and thereby confirm that the major binding site for SNX17 is confined to the NPXF2 motif in KRIT1. Taken together, our results verify a direct interaction between SNX17 and KRIT1 and classify KRIT1 as a SNX17 binding partner.     

Human hypoxanthine-guanine phosphoribosyltransferase: a single nucleotide substitution in cDNA clones isolated from a patient with Lesch-Nyhan syndrome (HPRTMidland)

We have determined the molecular basis for hypoxanthine-guanine phosphoribosyltransferase (HPRT) deficiency in a patient, J.H., with Lesch-Nyhan syndrome. Radioimmunoassay of lysates of erythrocytes or cultured B-lymphoblasts showed that this patient had no detectable HPRT enzyme activity or HPRT protein. HPRT-specific mRNA levels were normal by Northern analysis. We created a cDNA library from mRNA isolated from cultured lymphoblasts derived from this patient. Nucleotide sequencing of full-length HPRT cDNA clones revealed a single nucleotide (nt) substitution: a T-to-A transversion at nt 389. We have designated this variant HPRTMidland. The predicted amino acid (aa) substitution in HPRTMidland is a valine to aspartic acid at aa 130. This substitution is within 2 aa of the amino acid substitution in a previously defined HPRT variant, HPRTAnn Arbor. Both mutations are within a highly conserved sequence in the putative 5-phosphoribosyl-1-pyrophosphate-binding domain. The amino acid substitution in HPRTMidland causes a significant perturbation in the predicted secondary structure of this region. The HPRTMidland mutation affects a different domain of HPRT than the HPRTFlint mutation located at 167 nt away.     

Structural Determinants Allowing Endolysosomal Sorting and Degradation of Endosomal GTPases

Rapid control of protein degradation is usually achieved through the ubiquitin‐proteasome pathway. We recently found that the short‐lived GTPase RhoB is degraded in lysosomes. Moreover, the fusion of the RhoB C‐terminal sequence CINCCKVL, containing the isoprenylation and palmitoylation sites, to other proteins directs their sorting into multivesicular bodies (MVBs) and rapid lysosomal degradation. Here, we show that this process is highly specific for RhoB. Alteration of late endosome lipid dynamics produced the accumulation of RhoB, but not of other endosomal GTPases, including Rab5, Rab7, Rab9 or Rab11, into enlarged MVB. Other isoprenylated and bipalmitoylated GTPases, such as H‐Ras, Rap2A, Rap2B and TC10, were not accumulated into MVB and were stable. Remarkably, although TC10, which is highly homologous to RhoB, was stable, a sequence derived from its C‐terminus (CINCCLIT) elicited MVB sorting and degradation of a green fluorescent protein (GFP)‐chimeric protein. This led us to identify a cluster of basic amino acids (KKH) in the TC10 hypervariable region, constituting a secondary signal potentially involved in electrostatic interactions with membrane lipids. Mutation of this cluster allowed TC10 MVB sorting and degradation, whereas inserting it into RhoB hypervariable region rescued this protein from its lysosomal degradation pathway. These findings define a highly specific structural module for entering the MVB pathway and rapid lysosomal degradation.     

Single-cysteine substitution mutants at amino acid positions 306-321 in rhodopsin, the sequence between the cytoplasmic end of helix VII and the palmitoylation sites: sulfhydryl reactivity and transducin activation reveal a tertiary structure.

As sensors for structure at the cytoplasmic face of rhodopsin, single-cysteine substitution mutants have been previously studied in the regions connecting helices III and IV and helices V and VI. In this paper we report on single-cysteine substitution mutants at amino acid positions 306-321, comprising the cytoplasmic sequence between helix VII and the palmitoylation sites in rhodopsin. The cysteine opsin mutants were expressed in COS-1 cells and on treatment with 11-cis-retinal all formed the characteristic rhodopsin chromophore. Cysteines at positions 306-316 and 319 reacted in the dark with the thiol-specific reagent 4, 4'-dithiodipyridine (4-PDS) but showed a wide variation in reactivity. Cysteines at positions 317, 318, 320, and 321 showed no reaction with 4-PDS. The mutants on illumination also showed wide variations in activating GT. The mutant Y306C showed almost no GT activation, I307C and N310C were poor, and the activity of the mutants M309C, F313C, and M317C was also reduced relative to WT. The results suggest that the region comprising amino acids 306-321 is a part of a tertiary structure and that specific amino acids in this region on light-activation participate in the interaction with GT.     

Numb3 is an endocytosis adaptor for the inflammatory marker P-selectin

The endocytic protein Numb3 was found to bind to the cytosolic tail of the leukocyte adhesion receptor P-selectin. The N-terminal phosphotyrosine-binding (PTB) domain of Numb3 is responsible for this activity. An alanine scan revealed the FTNAAFD sequence as recognition region in P-selectin. Structural modeling of the interaction between the Numb PTB domain and the P-selectin tail suggests that both phenylalanines within the recognition sequence fit into hydrophobic cavities of the PTB surface. Their exchange for alanine gave Numb-negative mutants detaining the inhibition of P-selectin endocytosis by Numb PTB overexpression. Cells stable expressing P-selectins internalized the negative mutants markedly slower than the wild type. Consistent with other reports on the phosphorylation of Numb, we found that only the dephospho-Numb is able to bind P-selectin. Our observations demonstrate that Numb3 is an endocytic receptor for P-selectin and may be responsible for the rapid internalization of P-selectin when endothelial activation ends.     

Sorting of encystation-specific cysteine protease to lysosome-like peripheral vacuoles in Giardia lamblia requires a conserved tyrosine-based motif

Encystation-specific cysteine protease (ESCP) was the first membrane-associated protein described to be part of the lysosome-like peripheral vacuoles in the intestinal parasite Giardia lamblia. ESCP is homologous to cathepsin C enzymes of higher eukaryotes, but is distinguished from other lysosomal cysteine proteases because it possesses a transmembrane domain and a short cytoplasmic tail. Tyrosine-based motifs within tails of membrane proteins are known to participate in endosomal/lysosomal protein sorting in higher eukaryotes. In this study, we show that a YRPI motif within the ESCP cytoplasmic tail is necessary and sufficient to mediate ESCP sorting to peripheral vacuoles in Giardia. Deletion and point mutation analysis demonstrated that the tyrosine residue is critical for ESCP sorting, whereas amino acids located at the Y+1 (Arg), Y+2 (Pro), and Y+3 (Ile) positions show minimal effect. Loss of the motif resulted in surface localization, whereas addition of the motif to a variant-specific surface protein resulted in lysosomal localization. Although Giardia trophozoites lack a morphologically discernible Golgi apparatus, our findings indicate that this parasite directs proteins to the lysosomes using a conserved sorting signal similar to that used by yeast and mammalian cells. Because Giardia is one of the earliest branching protist, these results demonstrate that sorting motifs for specific protein traffic developed very early during eukaryotic evolution.     

A tyrosine motif in the cytoplasmic domain of mason-pfizer monkey virus is essential for the incorporation of glycoprotein into virions

Mason-Pfizer monkey virus (M-PMV) encodes a transmembrane (TM) glycoprotein with a 38-amino-acid-long cytoplasmic domain. After the release of the immature virus, a viral protease-mediated cleavage occurs within the cytoplasmic domain, resulting in the loss of 17 amino acids from the carboxy terminus. This maturational cleavage occurs between a histidine at position 21 and a tyrosine at position 22 in the cytoplasmic domain of the TM protein. We have demonstrated previously that a truncated TM glycoprotein with a 21-amino-acid-long cytoplasmic tail showed enhanced fusogenicity but could not be incorporated into virions. These results suggest that postassembly cleavage of the cytoplasmic domain removes a necessary incorporation signal and activates fusion activity. To investigate the contribution of tyrosine residues to the function of the glycoprotein complex and virus replication, we have introduced amino acid substitutions into two tyrosine residues found in the cytoplasmic domain. The effects of these mutations on glycoprotein biosynthesis and function, as well as on virus infectivity, have been examined. Mutation of tyrosine 34 to alanine had little effect on glycoprotein function. In contrast, substitutions at tyrosine 22 modulated fusion activity in either a positive or negative manner, depending on the substituting amino acid. Moreover, any nonaromatic substitution at this position blocked glycoprotein incorporation into virions and abolished infectivity. These results demonstrate that M-PMV employs a tyrosine signal for the selective incorporation of glycoprotein into budding virions. Antibody uptake studies show that tyrosine 22 is part of an efficient internalization signal in the cytoplasmic domain of the M-PMV glycoprotein that can also be positively and negatively influenced by changes at this site.     

Human apolipoprotein E mRNA. cDNA cloning and nucleotide sequencing of a new variant

The complete nucleotide sequences of three cloned cDNAs corresponding to human liver apolipoprotein E (apo-E) mRNA were determined. Analysis of the longest cDNA showed that it contained 1157 nucleotides of mRNA sequence with a 5'-terminal nontranslated region of 61 nucleotides, a signal peptide region corresponding to 18 amino acids, a mature protein region corresponding to 299 amino acids, and a 3'-terminal nontranslated region of 142 nucleotides. The inferred amino acid sequences from two cDNAs were identical and corresponded to the amino acid sequence for plasma apo-E3 that has been reported previously ( Rall , S. C., Jr., Weisgraber , K. H., and Mahley , R. W. (1982) J. Biol. Chem. 257, 4171-4178). The third cDNA differed from the other two cDNAs in five nucleotide positions. Three of these differences occurred in the third nucleotide position of amino acid codons, resulting in no change in the corresponding amino acids at residues Val-85, Ser-223, and Gln-248. The other two altered nucleotides occurred in the first nucleotide position of codons, leading to changes in the amino acids encoded. In the variant sequence, a threonine replaced the normal alanine at residue 99 and a proline replaced the normal alanine at residue 152. We have concluded that the human liver donor was heterozygous for the epsilon 3 genotype. The variant cDNA corresponds to a new, previously undescribed variant form of apo-E in which the amino acid substitutions of the protein are electrophoretically silent; it would probably be undetectable by standard apo-E phenotyping methods. The amino acid substitution at position 152 occurs in a region of apo-E that appears to be important for receptor binding, and it may have clinical significance.