Molecular cloning and further characterization of a probable plant vacuolar sorting receptor.

BP-80 is a type I integral membrane protein abundant in pea (Pisum sativum) clathrin-coated vesicles (CCVs) that binds with high affinity to vacuole-targeting determinants containing asparagine-proline-isoleucine-arginine. Here we present results from cDNA cloning and studies of its intracellular localization. Its sequence and sequences of homologs from Arabidopsis, rice (Oryza sativa), and maize (Zea mays) define a novel family of proteins unique to plants that is highly conserved in both monocotyledons and dicotyledons. The BP-80 protein is present in dilated ends of Golgi cisternae and in "prevacuoles," which are small vacuoles separate from but capable of fusing with lytic vacuoles. Its cytoplasmic tail contains a Tyr-X-X-hydrophobic residue motif associated with transmembrane proteins incorporated into CCVs. When transiently expressed in tobacco (Nicotiana tabacum) suspension-culture protoplasts, a truncated form lacking transmembrane and cytoplasmic domains was secreted. These results, coupled with previous studies of ligand-binding specificity and pH dependence, strongly support our hypothesis that BP-80 is a vacuolar sorting receptor that trafficks in CCVs between Golgi and a newly described prevacuolar compartment.     

Yeast Kex1p is a Golgi-associated membrane protein: deletions in a cytoplasmic targeting domain result in mislocalization to the vacuolar membrane

We have investigated the localization of Kex1p, a type I transmembrane carboxypeptidase involved in precursor processing within the yeast secretory pathway. Indirect immunofluorescence demonstrated the presence of Kex1p in a punctate organelle resembling the yeast Golgi apparatus as identified by Kex2p and Sec7p (Franzusoff, A., K. Redding, J. Crosby, R. S. Fuller, and R. Schekman. 1991. J. Cell Biol. 112:27-37). Glycosylation studies of Kex1p were consistent with a Golgi location, as Kex1p was progressively N-glycosylated in an MNN1-dependent manner. To address the basis of Kex1p targeting to the Golgi apparatus, we examined the cellular location of a series of carboxy-terminal truncations of the protein. The results indicate that a cytoplasmically exposed carboxy-terminal domain is required for retention of this membrane protein within the Golgi apparatus. Deletions of the retention region or overproduction of wild-type Kex1p led to mislocalization of Kex1p to the vacuolar membrane. This unexpected finding is discussed in terms of models involving either the vacuole as a default destination for membrane proteins, or by endocytosis to the vacuole following their default localization to the plasma membrane.     

Vacuolar storage proteins and the putative vacuolar sorting receptor BP-80 exit the golgi apparatus of developing pea cotyledons in different transport vesicles

In the parenchyma cells of developing legume cotyledons, storage proteins are deposited in a special type of vacuole, known as the protein storage vacuole (PSV). Storage proteins are synthesized at the endoplasmic reticulum and pass through the Golgi apparatus. In contrast to lysosomal acid hydrolases, storage proteins exit the Golgi apparatus in 130-nm-diameter electron-dense vesicles rather than in clathrin-coated vesicles. By combining isopycnic and rate zonal sucrose density gradient centrifugation with phase partitioning, we obtained a highly enriched dense vesicle fraction. This fraction contained prolegumin, which is the precursor of one of the major storage proteins. In dense vesicles, prolegumin occurred in a more aggregated form than it did in the endoplasmic reticulum. The putative vacuolar sorting receptor BP-80 was highly enriched in purified clathrin-coated vesicles, which, in turn, did not contain prolegumin. The amount of BP-80 was markedly reduced in the dense vesicle fraction. This result was confirmed by quantitative immunogold labeling of cryosections of pea cotyledons: whereas antibodies raised against BP-80 significantly labeled the Golgi stacks, labeling of the dense vesicles could not be detected. In contrast, 90% of the dense vesicles were labeled with antibodies raised against alpha-TIP (for tonoplast intrinsic protein), which is the aquaporin specific for the membrane of the PSV. These results lead to the conclusions that storage proteins and alpha-TIP are delivered via the same vesicular pathway into the PSVs and that the dense vesicles that carry these proteins in turn do not contain BP-80.     

The phosphate carrier has an ability to be sorted to either the TIM22 pathway or the TIM23 pathway for its import into yeast mitochondria

Most mitochondrial proteins are synthesized in the cytosol, imported into mitochondria via the TOM40 (translocase of the mitochondrial outer membrane 40) complex, and follow several distinct sorting pathways to reach their destination submitochondrial compartments. Phosphate carrier (PiC) is an inner membrane protein with 6 transmembrane segments (TM1-TM6) and requires, after translocation across the outer membrane, the Tim9-Tim10 complex and the TIM22 complex to be inserted into the inner membrane. Here we analyzed an in vitro import of fusion proteins between various PiC segments and mouse dihydrofolate reductase. The fusion protein without TM1 and TM2 was translocated across the outer membrane but was not inserted into the inner membrane. The fusion proteins without TM1-TM4 were not inserted into the inner membrane but instead translocated across the inner membrane. Functional defects of Tim50 of the TIM23 complex caused either by depletion of the protein or the addition of anti-Tim50 antibodies blocked translocation of the fusion proteins without TM1-TM4 across the inner membrane, suggesting that lack of TM1-TM4 led to switch of its sorting pathway from the TIM22 pathway to the TIM23 pathway. PiC thus appears to have a latent signal for sorting to the TIM23 pathway, which is exposed by reduced interactions with the Tim9-Tim10 complex and maintenance of the import competence.     

Lysosome associated membrane protein 1 (Lamp1) traffics directly from the TGN to early endosomes

The precise trafficking routes followed by newly synthesized lysosomal membrane proteins after exit from the Golgi are unclear. To study these events we created a novel chimera (YAL) having a lumenal domain comprising two tyrosine sulfation motifs fused to avidin, and the transmembrane and cytoplasmic domains of lysosome associated membrane protein 1 (Lamp1). The newly synthesized protein rapidly transited from the trans- Golgi Network (TGN) to lysosomes (t(1/2) approximately 30 min after a lag of 15-20 min). However, labeled chimera was captured by biotinylated probes endocytosed for only 5 min, indicating that the initial site of entry into the endocytic pathway was early endosomes. Capture required export of YAL from the TGN, and endocytosis of the biotinylated reagent, and was essentially quantitative within 2 h of chase, suggesting that all molecules were following an identical route. There was no evidence of YAL trafficking via the cell surface. Fusion of TGN-derived vesicles with 5 min endosomes could be recapitulated in vitro, but neither late endosomes nor lysosomes could serve as acceptor compartments. This suggests that contrary to previous conclusions, most if not all newly synthesized Lamp1 traffics from the TGN to early endosomes prior to delivery to late endosomes and lysosomes.     

Protein sorting to the vacuolar membrane.

The vacuolar membrane (tonoplast) of plant cells contains a polytopic integral membrane protein with six membrane-spanning domains and cytoplasmically oriented amino-terminal and carboxy-terminal domains. This protein, tonoplast intrinsic protein (TIP), is a member of the membrane intrinsic protein (MIP) family of proteins, a family of channel proteins found in a variety of organisms. In bean seeds, alpha-TIP is synthesized on the rough endoplasmic reticulum and its transport to the tonoplast is mediated by the secretory system. In this study, we report that a polypeptide segment that includes the sixth membrane domain and the cytoplasmic tail of 18 amino acids of alpha-TIP is sufficient to target the reporter protein phosphinotricine acetyltransferase to the tonoplast of stably transformed tobacco cells. To determine if the carboxy-terminal cytoplasmic tail of alpha-TIP contains important tonoplast targeting information, a deletion construct lacking the 15 carboxy-terminal amino acids was introduced for transient expression in tobacco cells; we found that the slightly truncated protein still accumulated in the tonoplast. From these results, we concluded that a transmembrane domain of a tonoplast protein probably contains sufficient information for transport to the tonoplast. Whether such transport occurs by bulk flow or involves specific cellular machinery remains to be determined.     

Integral Membrane Protein Sorting to Vacuoles in Plant Cells: Evidence for Two Pathways

Plant cells may contain two functionally distinct vacuolar compartments. Membranes of protein storage vacuoles (PSV) are marked by the presence of α-tonoplast intrinsic protein (TIP), whereas lytic vacuoles (LV) are marked by the presence of γ-TIP. Mechanisms for sorting integral membrane proteins to the different vacuoles have not been elucidated. Here we study a chimeric integral membrane reporter protein expressed in tobacco suspension culture protoplasts whose traffic was assessed biochemically by following acquisition of complex Asn-linked glycan modifications and proteolytic processing, and whose intracellular localization was determined with confocal immunofluorescence. We show that the transmembrane domain of the plant vacuolar sorting receptor BP-80 directs the reporter protein via the Golgi to the LV prevacuolar compartment, and attaching the cytoplasmic tail (CT) of γ-TIP did not alter this traffic. In contrast, the α-TIP CT prevented traffic of the reporter protein through the Golgi and caused it to be localized in organelles separate from ER and from Golgi and LV prevacuolar compartment markers. These organelles had a buoyant density consistent with vacuoles, and α-TIP protein colocalized in them with the α-TIP CT reporter protein when the two were expressed together in protoplasts. These results are consistent with two separate pathways to vacuoles for membrane proteins: a direct ER to PSV pathway, and a separate pathway via the Golgi to the LV.     

A vacuolar sorting receptor PV72 on the membrane of vesicles that accumulate precursors of seed storage proteins (PAC vesicles)

A novel vesicle, referred to as a precursor-accumulating (PAC) vesicle, mediates the transport of storage protein precursors to protein storage vacuoles in maturing pumpkin seeds. PV72, a type I integral membrane protein with three repeats of epidermal growth factor, was found on the membrane of the PAC vesicles. PV72 had an ability to bind to pro2S albumin, a storage protein precursor, in a Ca(2+)-dependent manner, via the C-terminal region of pro2S albumin, which was found to function as a vacuolar targeting signal. This implies that PV72 is a vacuolar sorting receptor of the storage protein. PV72 was specifically and transiently accumulated at the middle stage of seed maturation in association with the synthesis of storage proteins. Subcellular fractionation showed that PV72 was also accumulated in the microsomal fraction. A fusion protein consisting of GFP and the transmembrane domain and the cytosolic tail of PV72 was localized in Golgi complex. PV72 in the isolated PAC vesicles had a complex type of oligosaccharide, indicating that PV72 passed though the Golgi complex. These results suggest that PV72 is recycled between PAC vesicles and Golgi complex/post-Golgi compartments. PV72 appears to be responsible for recruiting pro2S albumin molecules from the Golgi complex to the PAC vesicles.     

Vacuolar storage proteins are sorted in the cis-cisternae of the pea cotyledon Golgi apparatus

Developing pea cotyledons contain functionally different vacuoles, a protein storage vacuole and a lytic vacuole. Lumenal as well as membrane proteins of the protein storage vacuole exit the Golgi apparatus in dense vesicles rather than in clathrin-coated vesicles (CCVs). Although the sorting receptor for vacuolar hydrolases BP-80 is present in CCVs, it is not detectable in dense vesicles. To localize these different vacuolar sorting events in the Golgi, we have compared the distribution of vacuolar storage proteins and of alpha-TIP, a membrane protein of the protein storage vacuole, with the distribution of the vacuolar sorting receptor BP-80 across the Golgi stack. Analysis of immunogold labeling from cryosections and from high pressure frozen samples has revealed a steep gradient in the distribution of the storage proteins within the Golgi stack. Intense labeling for storage proteins was registered for the cis-cisternae, contrasting with very low labeling for these antigens in the trans-cisternae. The distribution of BP-80 was the reverse, showing a peak in the trans-Golgi network with very low labeling of the cis-cisternae. These results indicate a spatial separation of different vacuolar sorting events in the Golgi apparatus of developing pea cotyledons.     

BP-80 and homologs are concentrated on post-Golgi,probable lytic prevacuolar compartments

Prevacuolar compartments (PVCs) are membrane-bound organelles that mediate protein traffic between Golgi and vacuoles in the plant secretory pathway. Here we identify and define organelles as the lytic prevacuolar compartments in pea and tobacco cells using confocal immunofluorescence. We use five different antibodies specific for a vacuolar sorting receptor (VSR) BP-80 and its homologs to detect the location of VSR proteins. In addition, we use well-established Golgi-markers to identify Golgi organelles. We further compare VSR-labeled organelles to Golgi organelles so that the relative proportion of VSR proteins in Golgi vs. PVCs can be quantitated. More than 90% of the BP-80-marked organelles are separate from Golgi organelles; thus, BP-80 and its homologs are predominantly concentrated on the lytic PVCs. Additionally, organelles marked by anti-AtPep12p (AtSYP21p) and anti-AtELP antibodies are also largely separate from Golgi apparatus, whereas VSR and AtPep12p (AtSYP21p) were largely colocalized. We have thus demonstrated in plant cells that VSR proteins are predominantly present in the lytic PVCs and have provided additional markers for defining plant PVCs using confocal immunofluorescence. Additionally, our approach will provide a rapid comparison between markers to quantitate protein distribution among various organelles.     

Targeting of a Nicotiana plumbaginifolia H+ -ATPase to the plasma membrane is not by default and requires cytosolic structural determinants

The structural determinants involved in the targeting of multitransmembrane-span proteins to the plasma membrane (PM) remain poorly understood. The plasma membrane H+ -ATPase (PMA) from Nicotiana plumbaginifolia, a well-characterized 10 transmembrane-span enzyme, was used as a model to identify structural elements essential for targeting to the PM. When PMA2 and PMA4, representatives of the two main PMA subfamilies, were fused to green fluorescent protein (GFP), the chimeras were shown to be still functional and to be correctly and rapidly targeted to the PM in transgenic tobacco. By contrast, chimeric proteins containing various combinations of PMA transmembrane spanning domains accumulated in the Golgi apparatus and not in the PM and displayed slow traffic properties through the secretory pathway. Individual deletion of three of the four cytosolic domains did not prevent PM targeting, but deletion of the large loop or of its nucleotide binding domain resulted in GFP fluorescence accumulating exclusively in the endoplasmic reticulum. The results show that, at least for this polytopic protein, the PM is not the default pathway and that, in contrast with single-pass membrane proteins, cytosolic structural determinants are required for correct targeting.     

Trafficking of green fluorescent protein-tagged SNARE proteins in HSY cells

SNARE proteins are widely accepted to be involved in the docking and fusion process of intracellular vesicle trafficking. VAMP-2, syntaxin-4, and SNAP-23 are plausible candidate SNARE proteins for non-neuronal exocytosis. Thus, we examined the localization, protein-protein interaction, and intracellular trafficking of these proteins by expressing them as green fluorescent protein (GFP)- and FLAG-tagged fusion proteins in various cells, including HSY cells, a human parotid epithelial cell line. GFP-VAMP-2 was ex-pressed strongly in the Golgi area and weakly on the plasma membrane. Although GFP-SNAP-23 seemed to be expressed universally in the cytosol, the GFP signal was clearly seen on the plasma membrane, when soluble GFP-SNAP-23 was removed by treatment with saponin. GFP-syntaxin-4 was undetectable on the plasma membrane but was strongly expressed on unidentified unusually large vesicles. GFP-syntaxin-4 without its transmembrane domain was still incompletely soluble and observed as aggregates. When syntaxin-4 and munc18c were coexpressed, syntaxin-4 was translocated at least in part to the plasma membrane. The protein-protein interaction between syntaxin-4 and VAMP-2 with their transmembrane domains was markedly inhibited on coexpression of munc18c. These results suggest that munc18c plays an important role in the trafficking of syntaxin-4 to its proper destination by preventing premature interactions with other proteins, including SNARE proteins.     

Localization of green fluorescent protein fusions with the seven Arabidopsis vacuolar sorting receptors to prevacuolar compartments in tobacco BY-2 cells

We have previously demonstrated that vacuolar sorting receptor (VSR) proteins are concentrated on prevacuolar compartments (PVCs) in plant cells. PVCs in tobacco (Nicotiana tabacum) BY-2 cells are multivesicular bodies (MVBs) as defined by VSR proteins and the BP-80 reporter, where the transmembrane domain (TMD) and cytoplasmic tail (CT) sequences of BP-80 are sufficient and specific for correct targeting of the reporter to PVCs. The genome of Arabidopsis (Arabidopsis thaliana) contains seven VSR proteins, but little is known about their individual subcellular localization and function. Here, we study the subcellular localization of the seven Arabidopsis VSR proteins (AtVSR1-7) based on the previously proven hypothesis that the TMD and CT sequences correctly target individual VSR to its final destination in transgenic tobacco BY-2 cells. Toward this goal, we have generated seven chimeric constructs containing signal peptide (sp) linked to green fluorescent protein (GFP) and TMD/CT sequences (sp-GFP-TMD/CT) of the seven individual AtVSR. Transgenic tobacco BY-2 cell lines expressing these seven sp-GFP-TMD-CT fusions all exhibited typical punctate signals colocalizing with VSR proteins by confocal immunofluorescence. In addition, wortmannin caused the GFP-marked prevacuolar organelles to form small vacuoles, and VSR antibodies labeled these enlarged MVBs in transgenic BY-2 cells. Wortmannin also caused VSR-marked PVCs to vacuolate in other cell types, including Arabidopsis, rice (Oryza sativa), pea (Pisum sativum), and mung bean (Vigna radiata). Therefore, the seven AtVSRs are localized to MVBs in tobacco BY-2 cells, and wortmannin-induced vacuolation of PVCs is a general response in plants.     

An essential cytoplasmic domain for the Golgi localization of coiled-coil proteins with a COOH-terminal membrane anchor

Giantin is a resident Golgi protein that has an extremely long cytoplasmic domain (about 370 kDa) and is anchored to the Golgi membrane by the COOH-terminal membrane-anchoring domain (CMD) with no luminal extension. We examined the essential domain of giantin required for Golgi localization by mutational analysis. The Golgi localization of giantin was not affected by the deletion of its CMD or by substitution with the CMD of syntaxin-2, a plasma membrane protein. The giantin CMD fused to the cytoplasmic domain of syntaxin-2 could not retain the chimera in the Golgi apparatus. Sequential deletion analysis showed that the COOH-terminal sequence (positions 3059--3161) adjacent to the CMD was the essential domain required for the Golgi localization of giantin. We also examined two other Golgi-resident proteins, golgin-84 and syntaxin-5, with a similar membrane topology as giantin. It was confirmed that the cytoplasmic domain of about 100 residues adjacent to the CMD was required for their Golgi localization. Taken together, these results suggest that the COOH-terminally anchored Golgi proteins with long cytoplasmic extensions have the Golgi localization signal(s) in the cytoplasmic sequence adjacent to the CMD. This is in contrast to previous observations that a transmembrane domain is required for Golgi localization by other Golgi proteins transported from the endoplasmic reticulum.     

The signal sequence of exported protein-1 directs the green fluorescent protein to the parasitophorous vacuole of transfected malaria parasites

The malaria parasite, Plasmodium falciparum, spends part of its life cycle inside the erythrocytes of its human host. In the mature stages of intraerythrocytic growth, the parasite undertakes extensive remodeling of its adopted cellular home by exporting proteins beyond the confines of its own plasma membrane. To examine the signals involved in export of parasite proteins, we have prepared transfected parasites expressing a chimeric protein comprising the N-terminal region of the Plasmodium falciparum exported protein-1 appended to green fluorescent protein. The majority of the population of the chimeric protein appears to be correctly processed and trafficked to the parasitophorous vacuole, indicating that this is the default destination for protein secretion. Some of the protein is redirected to the parasite food vacuole and further degraded. Photobleaching studies reveal that the parasitophorous vacuole contains subcompartments that are only partially interconnected. Dual labeling with the lipid probe, BODIPY-TR-ceramide, reveals the presence of membrane-bound extensions that can bleb from the parasitophorous vacuole to produce double membrane-bound compartments. We also observed regions and extensions of the parasitophorous vacuole, where there is segregation of the lumenal chimera from the lipid components. These regions may represent sites for the sorting of proteins destined for the trafficking to sites beyond the parasitophorous vacuole membrane.     

The First Transmembrane Domain of Lipid Phosphatase SAC1 Promotes Golgi Localization

The lipid phosphatase Sac1 cycles between endoplasmic reticulum and cisternal Golgi compartments. In proliferating mammalian cells, a canonical dilysine motif at the C-terminus of Sac1 is required for coatomer complex-I (COP-I)-binding and continuous retrieval to the ER. Starvation triggers accumulation of Sac1 at the Golgi. The mechanism responsible for Golgi retention of Sac1 is unknown. Here we show that the first of the two transmembrane regions in human SAC1 (TM1) functions in Golgi localization. A minimal construct containing only TM1 and the adjacent flanking sequences is concentrated at the Golgi. Transplanting TM1 into transferrin receptor 2 (TfR2) induces Golgi accumulation of this normally plasma membrane and endosomal protein, indicating that TM1 is sufficient for Golgi localization. In addition, we determined that the N-terminal cytoplasmic domain of SAC1 also promotes Golgi localization, even when TM1 is mutated or absent. We conclude that the distribution of SAC1 within the Golgi is controlled via both passive membrane thickness-dependent partitioning of TM1 and a retention mechanism that requires the N-terminal cytoplasmic region.     

Targeting of a heterodimeric membrane protein complex to the Golgi: rubella virus E2 glycoprotein contains a transmembrane Golgi retention signal.

Rubella virus (RV) envelope glycoproteins, E2 and E1, form a heterodimeric complex that is targeted to medial/trans-Golgi cisternae. To identify the Golgi targeting signal(s) for the E2/E1 spike complex, we constructed chimeric proteins consisting of domains from RV glycoproteins and vesicular stomatitis virus (VSV) G protein. The location of the chimeric proteins in stably transfected Chinese hamster ovary cells was determined by immunofluorescence, immunoelectron microscopy, and by the extent of processing of their N-linked glycans. A trans-dominant Golgi retention signal was identified within the C-terminal region of E2. When the transmembrane (TM) and cytoplasmic (CT) domains of VSV G were replaced with those of RV E2, the hybrid protein (G-E2TMCT+) was retained in the Golgi. Transport of G-E2TMCT+ to the Golgi was rapid (t1/2 = 10-20 min). The G-E2TMCT+ protein was determined to be distal to or within the medial Golgi based on acquisition of endo H resistance but proximal to the trans-Golgi network since it lacked sialic acid. Deletion analysis revealed that only the TM domain of E2 was required for Golgi targeting. Although the cytoplasmic domain of E2 was not necessary for Golgi retention, it was required for efficient transport of VSV G-RV chimeras out of the endoplasmic reticulum. When assayed in sucrose velocity sedimentations gradients, the Golgi-retained G-E2TMCT+ protein behaved as a dimer. Unlike virtually all other Golgi targeting signals, the E2 TM domain does not contain any polar amino acids. The TM and CT domains of E1 were not required for targeting of E2 and E1 to the Golgi indicating that a heterodimer of two integral membrane proteins can be retained in the Golgi by a single retention signal.     

A specific transmembrane domain of a coronavirus E1 glycoprotein is required for its retention in the Golgi region

The E1 glycoprotein of the avian coronavirus infectious bronchitis virus contains a short, glycosylated amino-terminal domain, three membrane-spanning domains, and a long carboxy-terminal cytoplasmic domain. We show that E1 expressed from cDNA is targeted to the Golgi region, as it is in infected cells. E1 proteins with precise deletions of the first and second or the second and third membrane-spanning domains were glycosylated, thus suggesting that either the first or third transmembrane domain can function as an internal signal sequence. The mutant protein with only the first transmembrane domain accumulated intracellularly like the wild-type protein, but the mutant protein with only the third transmembrane domain was transported to the cell surface. This result suggests that information specifying accumulation in the Golgi region resides in the first transmembrane domain, and provides the first example of an intracellular membrane protein that is transported to the plasma membrane after deletion of a specific domain.     

Importance of Cys,Gln, and Tyr from the transmembrane domain of human alpha 3/4 fucosyltransferase III for its localization and sorting in the Golgi of baby hamster kidney cells

Human fucosyltransferase III (EC ) (FT3wt) is localized in the Golgi of baby hamster kidney cells and synthesizes Lewis determinants associated with cell adhesion events. Replacement of the amino acid residues from the transmembrane domain (TM) Cys-16, Gln-23, Cys-29, and Tyr-33 by Leu (FT3np) caused a shift in enzyme localization to the plasma membrane. The mislocalization caused a dramatic decrease in the amount of biosynthetic products of FT3wt, the Lewis determinants. Determination of the expression levels on the surface with mutants of the enzyme, where one, two, or three of these residues were replaced by Leu, suggested that Cys from the TM was required for the localization of FT3 in the Golgi. Furthermore, Cys-23 and Cys-29 mediated the formation of disulfide-bonded dimers but not higher molecular weight oligomers. In vitro reconstitution of intra-Golgi transport showed that FT3wt was incorporated into coatomer protein (COP) I vesicles, contrary to FT3np. These data suggested that Cys, Gln, and Tyr residues are important for FT3wt sorting into the transport vesicles possibly due to interactions with other membrane proteins.