Formation of crystalloid endoplasmic reticulum induced by expression of synaptotagmin lacking the conserved WHXL motif in the C terminus. Structural importance of the WHXL motif in the C2B domain

Synaptotagmin (Syt) is a family of type I membrane proteins that consists of a single transmembrane domain, a spacer domain, two Ca(2+)-binding C2 domains, and a short C terminus. We recently showed that deletion of the short C terminus (17 amino acids) of Syt IV prevented the Golgi localization of Syt IV proteins in PC12 cells and induced granular structures of various sizes in the cell body by an unknown mechanism (Fukuda, M., Ibata, K., and Mikoshiba, K. (2001) J. Neurochem. 77, 730-740). In this study we showed by electron microscopy that these structures are crystalloid endoplasmic reticulum (ER), analyzed the mechanism of its induction, and demonstrated that: (a) mutation or deletion of the evolutionarily conserved WHXL motif in the C terminus of the synaptotagmin family (Syt DeltaC) destabilizes the C2B domain structure (i.e. causes misfolding of the protein), probably by disrupting the formation of stable anti-parallel beta-sheets between the beta-1 and beta-8 strands of the C2B domain; (b) the resulting malfolded proteins accumulate in the ER rather than being transported to other membrane structures (e.g. the Golgi apparatus), with the malfolded proteins also inducing the expression of BiP (immunoglobulin binding protein), one of the ER stress proteins; and (c) the ERs in which the Syt DeltaC proteins have accumulated associate with each other as a result of oligomerization capacity of the synaptotagmin family, because the Syt IDeltaC mutant, which lacks oligomerization activity, cannot induce crystalloid ER. Our findings indicate that the conserved WHXL motif is important not only for protein interaction site but for proper folding of the C2B domain.     

Effect of forskolin on synaptotagmin IV protein trafficking in PC12 cells

Synaptotagmin IV (Syt IV) was originally described as an immediate early gene product induced by forskolin or membrane depolarization in PC12 cells; however, nothing is known about the subcellular localization and transport of the newly translated Syt IV protein in PC12 cells. In this study, we investigated the transport mechanism of Syt IV protein induced by forskolin and found that forskolin treatment dramatically increases the Syt IV protein level (approximately 10-fold, to a level comparable to that of Syt IX) and promotes the transport of Syt IV protein from the Golgi to the cell periphery by a microtubule-dependent motor(s). The expression levels and subcellular localizations of two major Syt isoforms (I and IX) in PC12 cells, on the other hand, were unaffected by such treatment. Immunoelectron microscopic analysis showed that some Syt IV signals are clearly associated with dense-core vesicles in forskolin-treated PC12 cells, although the majority of the Syt IV molecules at the cell periphery were present on clear vesicular structures other than dense-core vesicles. An N-terminal antibody-uptake experiment indicated that Syt IV-containing vesicles in forskolin-treated PC12 cells undergo Ca(2+)-dependent exocytosis, because uptake of the anti-Syt IV-N antibody from the culture medium was slightly, but significantly, increased after forskolin treatment. Our results indicate that forskolin (or the increased cAMP level) is important for the transport of the Syt IV protein from the Golgi to the cell periphery, but not sufficient for the sorting of all Syt IV molecules to mature dense-core vesicles.     

Mechanism of the SDS-resistant synaptotagmin clustering mediated by the cysteine cluster at the interface between the transmembrane and spacer domains

Synaptotagmin I (Syt I), a proposed major Ca(2+) sensor in the central nervous system, has been hypothesized as functioning in an oligomerized state during neurotransmitter release. We previously showed that Syts I, II, VII, and VIII form a stable SDS-resistant, beta-mercaptoethanol-insensitive, and Ca(2+)-independent oligomer surrounding the transmembrane domain (Fukuda, M., and Mikoshiba, K. (2000) J. Biol. Chem. 275, 28180-28185), but little is known about the molecular mechanism of the Ca(2+)-independent oligomerization by the synaptotagmin family. In this study, we analyzed the Ca(2+)-independent oligomerization properties of Syt I and found that it shows two distinct forms of self-oligomerization activity: stable SDS-resistant self-oligomerization activity and relatively unstable SDS-sensitive self-oligomerization activity. The former was found to be mediated by a post-translationally modified (i.e. fatty-acylated) cysteine (Cys) cluster (Cys-74, Cys-75, Cys-77, Cys-79, and Cys-82) at the interface between the transmembrane and spacer domains of Syt I. We also show that the number of Cys residues at the interface between the transmembrane and spacer domains determines the SDS- resistant oligomerizing capacity of each synaptotagmin isoform: Syt II, which contains seven Cys residues, showed the strongest SDS-resistant oligomerizing activity in the synaptotagmin family, whereas Syt XII, which has no Cys residues, did not form any SDS-resistant oligomers. The latter SDS-sensitive self-oligomerization of Syt I is mediated by the spacer domain, because deletion of the whole spacer domain, including the Cys cluster, abolished it, whereas a Syt I(CA) mutant carrying Cys to Ala substitutions still exhibited self-oligomerization. Based on these results, we propose that the oligomerization of the synaptotagmin family is regulated by two distinct mechanisms: the stable SDS-resistant oligomerization is mediated by the modified Cys cluster, whereas the relatively unstable (SDS-sensitive) oligomerization is mediated by the environment of the spacer domain.     

Calcium-dependent and -independent hetero-oligomerization in the synaptotagmin family

Synaptotagmins constitute a family of membrane proteins that are characterized by one transmembrane region and two C2 domains. Recent genetic and biochemical studies have indicated that oligomerization of synaptotagmin (Syt) I is important for expression of function during exocytosis of synaptic vesicles. However, little is known about hetero-oligomerization in the synaptotagmin family. In this study, we showed that the synaptotagmin family is a type I membrane protein (N(lumen)/C(cytoplasm)) by introducing an artificial N-glycosylation site at the N-terminal domain, and systematically examined all the possible combinations of hetero-oligomerization among synaptotagmin family proteins (Syts I-XI). We classified the synaptotagmin family into four distinct groups based on differences in Ca(2+)-dependent and -independent oligomerization activity. Group A Syts (III, V, VI, and X) form strong homo- and hetero-oligomers by disulfide bonds at an N-terminal cysteine motif irrespective of the presence of Ca(2+) [Fukuda, M., Kanno, E., and Mikoshiba, K. (1999) J. Biol. Chem. 274, 31421-31427]. Group B Syts (I, II, VIII, and XI) show moderate homo-oligomerization irrespective of the presence of Ca(2+). Group C synaptotagmins are characterized by weak Ca(2+)-dependent (Syts IX) or no homo-oligomerization activity (Syt IV). Syt VII (Group D) has unique Ca(2+)-dependent homo-oligomerization properties with EC(50) values of about 150 microM Ca(2+) [Fukuda, M., and Mikoshiba, K. (2000) J. Biol. Chem. 275, 28180-28185]. Syts IV, VIII, and XI did not show any apparent hetero-oligomerization activity, but some sets of synaptotagmin isoforms can hetero-oligomerize in a Ca(2+)-dependent and/or -independent manner. Our data suggest that Ca(2+)-dependent and -independent hetero-oligomerization of synaptotagmins may create a variety of Ca(2+)-sensors.     

Molecular cloning and characterization of human,rat, and mouse synaptotagmin XV

Synaptotagmin (Syt) constitutes a large family of putative membrane trafficking proteins that share a short extracellular domain, a single N-terminal transmembrane domain, and C-terminal tandem C2 domains. In this study, I identified and characterized a novel member of the Syt family (named Syt XV-a) in the mouse, the rat, and humans. Although Syt XV-a protein has a short hydrophobic region at the very end of the N terminus (i.e., lacks a putative extracellular domain), biochemical and cellular analyses have indicated that the short hydrophobic region (amino acids 5-22) is sufficient for producing type I membrane topology in cultured cells, the same as in other Syt family proteins. Unlike other Syt isoforms, however, the mouse and human Syt XV have an alternative splicing isoform that lacks the C-terminal portion of the C2B domain (named Syt XV-b). Since the expression of Syt XV-a/b mRNA was mainly found in non-neuronal tissues (e.g., lung and testis) and Syt XV-a C2 domains lack Ca(2+)-dependent phospholipid binding activity, Syt XV-a is classified as a non-neuronal, Ca(2+)-independent Syt.     

Nerve growth factor-dependent sorting of synaptotagmin IV protein to mature dense-core vesicles that undergo calcium-dependent exocytosis in PC12 cells

Synaptotagmin IV (Syt IV) is a fourth member of the Syt family and has been shown to regulate some forms of memory and learning by analysis of Syt IV null mutant mice (Ferguson, G. D., Anagnostaras, S. G., Silva, A. J., and Herschman, H. R. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 5598-5603). However, the involvement of Syt IV protein in vesicular trafficking and even its localization in secretory vesicles are still matters of controversy. Here we present several lines of evidence showing that the Syt IV protein in PC12 cells is normally localized in the Golgi or immature vesicles at the cell periphery and is sorted to fusion-competent mature dense-core vesicles in response to short nerve growth factor (NGF) stimulation. (i) In undifferentiated PC12 cells, Syt IV protein is mainly localized in the Golgi and small amounts are also present at the cell periphery, but according to the results of an immunocytochemical analysis, they do not colocalize with conventional secretory vesicle markers (Syt I, Syt IX, Rab3A, Rab27A, vesicle-associated membrane protein 2, and synaptophysin) at all. By contrast, limited colocalization of Syt IV protein with dense-core vesicle markers is found in the distal parts of the neurites of NGF-differentiated PC12 cells. (ii) Immunoelectron microscopy with highly specific anti-Syt IV antibody revealed that the Syt IV protein in undifferentiated PC12 cells is mainly present on the Golgi membranes and immature secretory vesicles, whereas after NGF stimulation Syt IV protein is also present on the mature dense-core vesicles. (iii) An N-terminal antibody-uptake experiment indicated that Syt IV-containing vesicles in the neurites of NGF-differentiated PC12 cells undergo Ca(2+)-dependent exocytosis, whereas no uptake of the anti-Syt IV-N antibody was observed in undifferentiated PC12 cells. Our results suggest that Syt IV is a stimulus (e.g. NGF)-dependent regulator for exocytosis of dense-core vesicles.     

A novel alternatively spliced variant of synaptotagmin VI lacking a transmembrane domain. Implications for distinct functions of the two isoforms.

Synaptotagmins are a family of membrane proteins that are characterized by a single transmembrane region and tandem C2 domains and that are likely to regulate constitutive and/or regulated vesicle traffic. We have shown that a subclass of synaptotagmins (III, V, VI, and X) forms homo- and heterodimers through an evolutionarily conserved cysteine motif at their N termini (Fukuda, M., Kanno, E., and Mikoshiba, K. (1999) J. Biol. Chem. 274, 31421-31427). In this study, we identified a novel alternatively spliced variant of synaptotagmin (Syt) VI that lacks the N-terminal 85 amino acids including the transmembrane region (thus designated as Syt VIDeltaTM). Because it lacks the cysteine motif responsible for self-dimerization, Syt VIDeltaTM could not associate with Syt VI even in the presence of Ca(2+). Despite lacking the transmembrane region, Syt VIDeltaTM can associate with the plasma membrane through the C-terminal 29 amino acids. In adult mouse brain, two closely comigrating bands at M(r) approximately 50,000, which closely corresponded to the molecular weight of recombinant Syt VIDeltaTM, were detected by anti-Syt VI antibody. These immunoreactive bands were found in both soluble and membrane fractions of mouse brain, indicating that they are membrane-associated proteins (Syt VIDeltaTM), but not transmembrane proteins (Syt VI). Expression of Syt VI and Syt VIDeltaTM in PC12 or COS-7 cells indicated that the two molecules have a distinct subcellular distribution: Syt VIDeltaTM is present in the cytosol or is associated with the plasma membrane or internal membrane structures, whereas Syt VI is localized to the endoplasmic reticulum and/or Golgi-like perinuclear compartment. These results suggest that Syt VI and Syt VIDeltaTM may play distinct roles in vesicular trafficking.     

Conserved N-terminal cysteine motif is essential for homo- and heterodimer formation of synaptotagmins III, V, VI, and X.

The synaptotagmins now constitute a large family of membrane proteins characterized by one transmembrane region and two C2 domains. Dimerization of synaptotagmin (Syt) I, a putative low affinity Ca(2+) sensor for neurotransmitter release, is thought to be important for expression of function during exocytosis of synaptic vesicles. However, little is known about the self-dimerization properties of other isoforms. In this study, we demonstrate that a subclass of synaptotagmins (III, V, VI, and X) (Ibata, K., Fukuda, M., and Mikoshiba, K. (1998) J. Biol. Chem. 273, 12267-12273) forms beta-mercaptoethanol-sensitive homodimers and identify three evolutionarily conserved cysteine residues at the N terminus (N-terminal cysteine motif, at amino acids 10, 21, and 33 of mouse Syt III) that are not conserved in other isoforms. Site-directed mutagenesis of these cysteine residues and co-immunoprecipitation experiments clearly indicate that the first cysteine residue is essential for the stable homodimer formation of Syt III, V, or VI, and heterodimer formation between Syts III, V, VI, and X. We also show that native Syt III from mouse brain forms a beta-mercaptoethanol-sensitive homodimer. Our results suggest that the cysteine-based heterodimerization between Syt III and Syt V, VI, or X, which have different biochemical properties, may modulate the proposed function of Syt III as a putative high affinity Ca(2+) sensor for neurotransmitter release.     

The Golgi-localized Arabidopsis endomembrane protein12 contains both endoplasmic reticulum export and Golgi retention signals at its C terminus

Endomembrane proteins (EMPs), belonging to the evolutionarily conserved transmembrane nine superfamily in yeast and mammalian cells, are characterized by the presence of a large lumenal N terminus, nine transmembrane domains, and a short cytoplasmic tail. The Arabidopsis thaliana genome contains 12 EMP members (EMP1 to EMP12), but little is known about their protein subcellular localization and function. Here, we studied the subcellular localization and targeting mechanism of EMP12 in Arabidopsis and demonstrated that (1) both endogenous EMP12 (detected by EMP12 antibodies) and green fluorescent protein (GFP)-EMP12 fusion localized to the Golgi apparatus in transgenic Arabidopsis plants; (2) GFP fusion at the C terminus of EMP12 caused mislocalization of EMP12-GFP to reach post-Golgi compartments and vacuoles for degradation in Arabidopsis cells; (3) the EMP12 cytoplasmic tail contained dual sorting signals (i.e., an endoplasmic reticulum export motif and a Golgi retention signal that interacted with COPII and COPI subunits, respectively); and (4) the Golgi retention motif of EMP12 retained several post-Golgi membrane proteins within the Golgi apparatus in gain-of-function analysis. These sorting signals are highly conserved in all plant EMP isoforms and, thus, likely represent a general mechanism for EMP targeting in plant cells.     

Synaptotagmin IV Acts as a Multi-Functional Regulator of Ca<Superscript>2+</Superscript>-Dependent Exocytosis

In response to stimuli, secretary cells secrete a variety of signaling molecules packed in vesicles (e.g., neurotransmitters and peptide hormones) into the extracellular space by exocytosis. The vesicle secretion is often triggered by calcium ion (Ca²⁺) entered into secretary cells and achieved by the fusion of secretory vesicles with the plasma membrane. Recent accumulating evidence has indicated that members of the synaptotagmin (Syt) family play a major role in Ca²⁺-dependent exocytosis, and Syt I, in particular, is now widely accepted as the major Ca²⁺-sensor for synchronous neurotransmitter release. Involvement of other Syt isoforms in Ca²⁺-dependent exocytotic events other than neurotransmitter release has also been reported, and the Syt IV isoform is of particular interest, because Syt IV has several unique features not found in Syt I (e.g., immediate early gene product induced by deporalization and postsynaptic localization). In this article, we summarize the literature on the multi-functional role of Syt IV in Ca²⁺-dependent exocytosis.     

Differential distribution of synaptotagmin-1, -4, -7, and -9 in rat adrenal chromaffin cells

Neurons and certain kinds of endocrine cells, such as adrenal chromaffin cells, have large dense-core vesicles (LDCVs) and synaptic vesicles or synaptic-like microvesicles (SLMVs). These secretory vesicles exhibit differences in Ca(2+) sensitivity and contain diverse signaling substances. The present work was undertaken to identify the synaptotagmin (Syt) isoforms present in secretory vesicles. Fractionation analysis of lysates of the bovine adrenal medulla and immunocytochemistry in rat chromaffin cells indicated that Syt 1 was localized in LDCVs and SLMVs, whereas Syt 7 was the predominant isoform present in LDCVs. In contrast to PC12 cells and the pancreatic β cell line INS-1, Syt 9 was not immunodetected in LDCVs in rat chromaffin cells. Double-staining revealed that Syt 9-like immunoreactivity was nearly identical with fluorescent thapsigargin binding, suggesting the presence of Syt 9 in the endoplasmic reticulum (ER).The exogenous expression of Syt 1-GFP in INS-1 cells, which had a negligible level of endogenous Syt 1, resulted in an increase in the amount of Syt 9 in the ER, suggesting that Syt 9 competes with Syt 1 for trafficking from the ER to the Golgi complex. We conclude that LDCVs mainly contain Syt 7, whereas SLMVs contain Syt 1, but not Syt 7, in rat and bovine chromaffin cells.     

Synaptotagmin VII is targeted to dense-core vesicles and regulates their Ca2+ -dependent exocytosis in PC12 cells

It has recently been proposed that synaptotagmin (Syt) VII functions as a plasma membrane Ca2+ sensor for dense-core vesicle exocytosis in PC12 cells based on the results of transient overexpression studies using green fluorescent protein (GFP)-tagged Syt VII; however, the precise subcellular localization of Syt VII is still a matter of controversy (plasma membrane versus secretory granules). In this study we established a PC12 cell line "stably expressing" the Syt VII-GFP molecule and demonstrated by immunocytochemical and immunoelectron microscopic analyses that the Syt VII-GFP protein is localized on dense-core vesicles as well as in other intracellular membranous structures, such as the trans-Golgi network and lysosomes. Syt VII-GFP forms a complex with endogenous Syts I and IX, but not with Syt IV, and it colocalize well with Syts I and IX in the cellular processes (where dense-core vesicles are accumulated) in the PC12 cell line. We further demonstrated by an N-terminal antibody-uptake experiment that Syt VII-GFP-containing dense-core vesicles undergo Ca2+ -dependent exocytosis, the same as endogenous Syt IX-containing vesicles. Moreover, silencing of Syt VII-GFP with specific small interfering RNA dramatically reduced high KCl-dependent neuropeptide Y secretion from the stable PC12 cell line (approximately 60% of the control cells), whereas the same small interfering RNA had little effect on neuropeptide Y secretion from the wild-type PC12 cells (approximately 85-90% of the control cells), indicating that the level of endogenous expression of Syt VII molecules must be low. Our results indicate that the targeting of Syt VII-GFP molecules to specific membrane compartment(s) is affected by the transfection method (transient expression versus stable expression) and suggested that Syt VII molecule on dense-core vesicles functions as a vesicular Ca2+ sensor for exocytosis in endocrine cells.     

Functional and Biochemical Analysis of the C2 Domains of Synaptotagmin IV

Synaptotagmins (Syts) are a family of vesicle proteins that have been implicated in both regulated neurosecretion and general membrane trafficking. Calcium-dependent interactions mediated through their C2 domains are proposed to contribute to the mechanism by which Syts trigger calcium-dependent neurotransmitter release. Syt IV is a novel member of the Syt family that is induced by cell depolarization and has a rapid rate of synthesis and a short half-life. Moreover, the C2A domain of Syt IV does not bind calcium. We have examined the biochemical and functional properties of the C2 domains of Syt IV. Consistent with its non-calcium binding properties, the C2A domain of Syt IV binds syntaxin isoforms in a calcium-independent manner. In neuroendocrine pheochromocytoma (PC12) cells, Syt IV colocalizes with Syt I in the tips of the neurites. Microinjection of the C2A domain reveals that calcium-independent interactions mediated through this domain of Syt IV inhibit calcium-mediated neurotransmitter release from PC12 cells. Conversely, the C2B domain of Syt IV contains calcium binding properties, which permit homo-oligomerization as well as hetero-oligomerization with Syt I. Our observation that different combinatorial interactions exist between Syt and syntaxin isoforms, coupled with the calcium stimulated hetero-oligomerization of Syt isoforms, suggests that the secretory machinery contains a vast repertoire of biochemical properties for sensing calcium and regulating neurotransmitter release accordingly.     

Distinct self-oligomerization activities of synaptotagmin family. Unique calcium-dependent oligomerization properties of synaptotagmin VII

Synaptotagmins constitute a large protein family, characterized by one transmembrane region and two C2 domains, and can be classified into several subclasses based on phylogenetic relationships and biochemical activities (Fukuda, M., Kanno, E., and Mikoshiba, K. (1999) J. Biol. Chem. 274, 31421-31427). Synaptotagmin I (Syt I), a possible Ca(2+) sensor for neurotransmitter release, showed both Ca(2+)-dependent (via the C2 domain) and -independent (via the NH(2)-terminal domain) self-oligomerization, which are thought to be important for synaptic vesicle exocytosis. However, little is known about the relationship between these two interactions and the Ca(2+)-dependent oligomerization properties of other synaptotagmin isoforms. In this study, we first examined the Ca(2+)-dependent self-oligomerization properties of synaptotagmin family by co-expression of T7- and FLAG-tagged Syts (full-length or cytoplasmic domain) in COS-7 cells. We found that Syt VII is a unique class of synaptotagmins that only showed robust Ca(2+)-dependent self-oligomerization at the cytoplasmic domain with EC(50) values of about 150 micrometer Ca(2+). In addition, Syt VII preferentially interacted with the previously described subclass of Syts (V, VI, and X) in a Ca(2+)-dependent manner. Co-expression of full-length and cytoplasmic portion of Syts VII (or II) indicate that Syt VII cytoplasmic domain oligomerizes in a Ca(2+)-dependent manner without being tethered at the NH(2)-terminal domain, whereas Ca(2+)-dependent self-oligomerization at the cytoplasmic domain of other isoforms (e.g. Syt II) occurs only when the two molecules are tethered at the NH(2)-terminal domain.     

Topological characterization of the essential Escherichia coli cell division protein FtsN.

Genetic and biochemical approaches were used to analyze a topological model for FtsN, a 36-kDa protein with a putative transmembrane segment near the N terminus, and to ascertain the requirements of the putative cytoplasmic and membrane-spanning domains for the function of this protein. Analysis of FtsN-PhoA fusions revealed that the putative transmembrane segment of FtsN could act as a translocation signal. Protease accessibility studies of FtsN in spheroblasts and inverted membrane vesicles confirmed that FtsN had a simple bitopic topology with a short cytoplasmic amino terminus, a single membrane-spanning domain, and a large periplasmic carboxy terminus. To ascertain the functional requirements of the N-terminal segments of FtsN, various constructs were made. Deletion of the N-terminal cytoplasmic and membrane-spanning domains led to intracellular localization of the carboxy domain, instability,and loss of function. Replacement of the N-terminal cytoplasmic and membrane-spanning domains with a membrane-spanning domain from MalG restored subcellular localization and function. These N-terminal domains of FtsN could also be replaced by the cleavable MalE signal sequence with restoration of subcellular localization and function. It is concluded that the N-terminal, cytoplasmic, and transmembrane domains of FtsN are not required for function of the carboxy domain other than to transport it to the periplasm. FtsQ and FtsI were also analyzed.     

Synaptotagmin I and IX function redundantly in controlling fusion pore of large dense core vesicles

Synaptotagmins (Syts) constitute a large family of at least 16 members and individual Syt isoforms exhibit distinct Ca²⁺-binding properties and subcellular localization. It remains to be demonstrated whether multiple Syt isoforms can function independently or cooperatively on certain type of vesicle. In the current study, we have developed NPY-pHluorin to specifically assess exocytosis of large dense core vesicles (LDCVs) and studied the requirement of Syt I and Syt IX for LDCV exocytosis in PC12 cells. We found that down-regulation of both Syt I and Syt IX resulted in a significant loss of Ca²⁺-dependent LDCV exocytosis. Moreover, our results suggest Syt I and Syt IX play redundant role in controlling the choice of fusion modes. Down-regulation of both Syt I and Syt IX renders more fusion in the kiss-and-run mode. We conclude that Syt I and Syt IX function redundantly in Ca²⁺-sensing and fusion pore dilation on LDCVs in PC12 cells.     

Localization and targeting of the Saccharomyces cerevisiae Kre2p/Mnt1p alpha 1,2-mannosyltransferase to a medial-Golgi compartment

The yeast Kre2p/Mnt1p alpha 1,2-mannosyltransferase is a type II membrane protein with a short cytoplasmic amino terminus, a membrane- spanning region, and a large catalytic luminal domain containing one N- glycosylation site. Anti-Kre2p/Mnt1p antibodies identify a 60-kD integral membrane protein that is progressively N-glycosylated in an MNN1-dependent manner. Kre2p/Mnt1p is localized in a Golgi compartment that overlaps with that containing the medial-Golgi mannosyltransferase Mnn1p, and distinct from that including the late Golgi protein Kex1p. To determine which regions of Kre2p/Mnt1p are required for Golgi localization, Kre2p/Mnt1p mutant proteins were assembled by substitution of Kre2p domains with equivalent sequences from the vacuolar proteins DPAP B and Pho8p. Chimeric proteins were tested for correct topology, in vitro and in vivo activity, and were localized intracellularly by indirect immunofluorescence. The results demonstrate that the NH2-terminal cytoplasmic domain is necessary for correct Kre2p Golgi localization whereas, the membrane-spanning and stem domains are dispensable. However, in a test of targeting sufficiency, the presence of the entire Kre2p cytoplasmic tail, plus the transmembrane domain and a 36-amino acid residue luminal stem region was required to localize a Pho8p reporter protein to the yeast Golgi.     

Kissing and pinching: synaptotagmin and calcium do more between bilayers

Building on recent findings that synaptotagmin (Syt) participates in synaptic vesicle endocytosis, Poskanzer et al., in this issue of Neuron, show distinct mechanisms by which Syt functions in this process. Most significantly, they show (1) that calcium binding to Syt determines the rate but not fidelity of vesicle recycling and (2) that mutations in a different Syt domain affect the shape but not rate of formation of recycled synaptic vesicles.     

Synaptotagmin-like protein 1-3: a novel family of C-terminal-type tandem C2 proteins

Synaptotagmins (Syt), rabphilin-3A, and Doc2 belong to a family of carboxyl terminal type (C-type) tandem C2 proteins and are thought to be involved in vesicular trafficking. We have cloned and characterized a novel family of C-type tandem C2 proteins, designated Slp1-3 (synaptotagmin-like protein 1-3). The Slp1-3 C2 domains show high homology to granuphilin-a C2 domains, but the amino-terminal domain of Slp1-3 does not contain any known protein motifs or a transmembrane domain. A subcellular fractionation study indicated that Slp1-3 proteins are peripheral membrane proteins. Phospholipid binding experiments indicated that Slp3 is a Ca(2+)-dependent isoform, but Slp1 and Slp2 are Ca(2+)-independent isoforms, because only the Slp3 C2A domain showed Ca(2+)-dependent phospholipid binding activity. The C-terminus of Slp1-3 also bound neurexin Ialpha in vitro, in the same manner as Syt family proteins, which may be important for the membrane association of Slp1-3. In addition, Slp family proteins are differentially distributed in different mouse tissues and at different developmental stages.     

Mutations of the Rous sarcoma virus env gene that affect the transport and subcellular location of the glycoprotein products

The envelope glycoproteins of Rous sarcoma virus (RSV), gp85 and gp37, are anchored in the membrane by a 27-amino acid, hydrophobic domain that lies adjacent to a 22-amino acid, cytoplasmic domain at the carboxy terminus of gp37. We have altered these cytoplasmic and transmembrane domains by introducing deletion mutations into the molecularly cloned sequences of a proviral env gene. The effects of the mutations on the transport and subcellular localization of the Rous sarcoma virus glycoproteins were examined in monkey (CV-1) cells using an SV40 expression vector. We found, on the one hand, that replacement of the nonconserved region of the cytoplasmic domain with a longer, unrelated sequence of amino acids (mutant C1) did not alter the rate of transport to the Golgi apparatus nor the appearance of the glycoprotein on the cell surface. Larger deletions, extending into the conserved region of the cytoplasmic domain (mutant C2), resulted in a slower rate of transport to the Golgi apparatus, but did not prevent transport to the cell surface. On the other hand, removal of the entire cytoplasmic and transmembrane domains (mutant C3) did block transport and therefore did not result in secretion of the truncated protein. Our results demonstrate that the C3 polypeptide was not transported to the Golgi apparatus, although it apparently remained in a soluble, nonanchored form in the lumen of the rough endoplasmic reticulum; therefore, it appears that this mutant protein lacks a functional sorting signal. Surprisingly, subcellular localization by internal immunofluorescence revealed that the C3 protein (unlike the wild type) did not accumulate on the nuclear membrane but rather in vesicles distributed throughout the cytoplasm. This observation suggests that the wild-type glycoproteins (and perhaps other membrane-bound or secreted proteins) are specifically transported to the nuclear membrane after their biosynthesis elsewhere in the rough endoplasmic reticulum.