scholarly journals Membrane protein sorting in the yeast secretory pathway: evidence that the vacuole may be the default compartment.

1992 ◽  
Vol 119 (1) ◽  
pp. 69-83 ◽  
Author(s):  
C J Roberts ◽  
S F Nothwehr ◽  
T H Stevens

The targeting signals of two yeast integral membrane dipeptidyl aminopeptidases (DPAPs), DPAP B and DPAP A, which reside in the vacuole and the Golgi apparatus, respectively, were analyzed. No single domain of DPAP B is required for delivery to the vacuolar membrane, because removal or replacement of either the cytoplasmic, transmembrane, or lumenal domain did not affect the protein's transport to the vacuole. DPAP A was localized by indirect immunofluorescence to non-vacuolar, punctate structures characteristic of the yeast Golgi apparatus. The 118-amino acid cytoplasmic domain of DPAP A is sufficient for retention of the protein in these structures, since replacement of the cytoplasmic domain of DPAP B with that of DPAP A resulted in an immunolocalization pattern indistinguishable from that of wild type DPAP A. Overproduction of DPAP A resulted in its mislocalization to the vacuole, because cells expressing high levels of DPAP A exhibited vacuolar as well as Golgi staining. Deletion of 22 residues of the DPAP A cytoplasmic domain resulted in mislocalization of the mutant protein to the vacuole. Thus, the cytoplasmic domain of DPAP A is both necessary and sufficient for Golgi retention, and removal of the retention signal, or saturation of the retention apparatus by overproducing DPAP A, resulted in transport to the vacuole. Like wild type DPAP B, the delivery of mutant membrane proteins to the vacuole was unaffected in the secretory vesicle-blocked sec1 mutant; thus, transport to the vacuole was not via the plasma membrane followed by endocytosis. These data are consistent with a model in which membrane proteins are delivered to the vacuole along a default pathway.

1984 ◽  
Vol 99 (6) ◽  
pp. 2011-2023 ◽  
Author(s):  
J W Wills ◽  
R V Srinivas ◽  
E Hunter

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.


1986 ◽  
Vol 6 (11) ◽  
pp. 3734-3745 ◽  
Author(s):  
H Shida

Two classes of revertants were isolated from a vaccinia virus mutant whose hemagglutinins (HAs) accumulate on nuclear envelopes and rough endoplasmic reticulums. The HAs of one of the revertants had the same phenotype as the wild type, i.e., rapid and efficient movement to the cell surface. The HAs of the second class had biphasic transport: rapid export to the cell surface as in the wild type and slow movement to the medial cisternae of the Golgi apparatus. Biochemical and nucleotide sequence analyses showed that the HAs of all the mutants examined that have defects in transport from the rough endoplasmic reticulum to the Golgi apparatus have altered cytoplasmic domains and that the HAs of the second class of revertants lack the whole cytoplasmic domain, while the HAs of the first class of revertants have a wild-type cytoplasmic domain.


2001 ◽  
Vol 114 (12) ◽  
pp. 2231-2239 ◽  
Author(s):  
Alain Rambourg ◽  
Catherine L. Jackson ◽  
Yves Clermont

The structural elements of the secretory pathway in the budding yeast Saccharomyces cerevisiae were analyzed by 3D stereo-electron microscopy using relatively thick sections in which membranes were selectively impregnated. In a wild-type strain, tubular networks of various sizes and staining properties were distributed throughout the cytoplasm. As a rule, wide-meshed, lightly stained polygonal networks were connected to more or less fenestrated sheets of endoplasmic reticulum (ER). Some of these networks were continuous with more intensely stained networks and narrower meshes that displayed at their intersections nodular dilations that progressively increased in size and staining properties to reach those of secretion granules. Such networks presumably corresponded to Golgi elements. Indeed, stacked cisternae typical of the mammalian Golgi apparatus are rarely found in wild-type cells. However, if it is assumed that the Golgi apparatus plays a key role in the segregation and maturation of secretion granules, then tubular networks with nodular dilations should be equivalent to parts of this organelle. In correlation with the increase in size and density of the nodules there was a decrease in diameter and staining intensity of the interconnecting tubules. These results parallel observations on the formation of secretory granules in mammalian cells and suggest that the segregation of secretory material is concomitant with the progressive perforation and tubulization of previously unperforated sheets. When the sec21-3 thermosensitive mutant was examined at the nonpermissive temperature (37°C), the secretory pathway was blocked at exit from the ER, which started to accumulate as clusters of narrow, anastomosed, unperforated ribbon-like elements. When the block was released by shifting down to permissive temperature (24°C), tubular networks of various sizes and caliber, presumably Golgi in nature, formed as soon as 5 minutes after release of the block. At later time intervals, granules of various sizes and densities appeared to be released by rupture of these tubular networks or even to form at the edges of ER fenestrae. These observations support a dynamic maturation process in which the formation of secretion granules occurs by means of an oriented series of membrane transformations starting at the ER and culminating with the liberation of secretion granules from Golgi networks.


1994 ◽  
Vol 302 (3) ◽  
pp. 641-648 ◽  
Author(s):  
R S McLeod ◽  
C Robbins ◽  
A Burns ◽  
Z Yao ◽  
P H Pritchard

Human apolipoprotein (apo) A-I is secreted as a proprotein of 249 amino acids and is processed extracellularly to the mature form (243 amino acids) by removal of a six-residue propeptide segment. We have examined the role of the apoA-I propeptide in intracellular transport and secretion using transfected baby hamster kidney cells that secreted either proapoA-I (from the wild-type cDNA, A-Iwt) or mature-form apoA-I (from A-I delta pro, a cDNA in which the propeptide sequence was deleted). Deletion of the propeptide from the apoA-I sequence did not affect the rate of apoA-I synthesis, nor did it affect the fidelity of proteolytic removal of the prepeptide. However, the propeptide deletion caused mature-form apoA-I to accumulate within the cells as determined by pulse-chase experiments; the intracellular retention times for the mature-form apoA-I in which the propeptide was prematurely removed was three times longer than that of proapoA-I (t1/2 > 3 h compared with approximately 50 min). There was no detectable degradation of either form of newly synthesized apoA-I. Immunofluorescence microscopy revealed that, whereas the proapoA-I was located predominantly in the Golgi apparatus, large quantities of the mature-form apoA-I were detected in the endoplasmic reticulum and very little was in the Golgi apparatus of A-I delta pro-transfected cells. These findings suggest that the propeptide sequence may be involved in the intracellular transport of apoA-I from the endoplasmic reticulum to the Golgi apparatus. We propose that the function of the propeptide sequence is to facilitate efficient transport of apoA-I through the secretory pathway.


1992 ◽  
Vol 3 (3) ◽  
pp. 309-321 ◽  
Author(s):  
M Disdier ◽  
J H Morrissey ◽  
R D Fugate ◽  
D F Bainton ◽  
R P McEver

P-selectin (CD62), formerly called GMP-140 or PADGEM, is a membrane protein located in secretory storage granules of platelets and endothelial cells. To study the mechanisms responsible for the targeting of P-selectin to storage granules, we transfected its cDNA into COS-7 and CHO-K1 cells, which lack a regulated exocytic pathway, or into AtT20 cells, which are capable of regulated secretion. P-selectin was expressed on the plasma membrane of COS-7 and CHO-K1 cells but was concentrated in storage granules of AtT20 cells. Immunogold electron microscopy indicated that the electron-dense granules containing P-selectin in AtT20 cells also stored the endogenous soluble hormone ACTH. Activation of AtT20 cells with 8-Br-cAMP increased the surface expression of P-selectin, consistent with agonist-induced fusion of granule membranes with the plasma membrane. Deletion of the last 23 amino acids of the 35-residue cytoplasmic domain resulted in delivery of P-selectin to the plasma membrane of AtT20 cells. Replacement of the cytoplasmic tail of tissue factor, a plasma membrane protein, with the cytoplasmic domain of P-selectin redirected the chimeric molecule to granules. We conclude that the cytoplasmic domain of P-selectin is both necessary and sufficient for sorting of membrane proteins into the regulated pathway of secretion.


2000 ◽  
Vol 113 (13) ◽  
pp. 2507-2516 ◽  
Author(s):  
G. Emery ◽  
M. Rojo ◽  
J. Gruenberg

Recent studies show that small trans-membrane proteins of approximately 22–24 kDa (the p24 family), which are grouped into 4 sub-families by sequence homology (p23, p24, p25 and p26), are involved in the early secretory pathway. In this study, we have investigated the mutual requirements of ectopically expressed members of the p24 family for targeting to their proper cellular destination. We find that coexpression of p23 and p24 is both necessary and sufficient for each protein to be transported to the cis-Golgi network/Golgi complex. Proteins from other subfamilies did not substitute for either p23 or p24, even after multiple coexpression. However, trafficking of the p23/p24 couple was facilitated by coexpression of proteins from other sub-families. In addition, we find that the sequence resembling an endoplasmic reticulum retrieval signal present in the cytoplasmic domain of p23 (but not p24) is dispensable. In contrast, the conserved coiled-coil region in the lumenal domain is absolutely required in both p23 and p24 for proper targeting of the p23/p24 couple. These data demonstrate that p23 and p24 must interact with each other to reach their destination, but that this strict requirement is combined with a mutual dependence amongst p24 proteins. We speculate that p24 proteins can form different oligomeric complexes, which contribute to confer specialized sorting/trafficking properties to membranes of the early secretory pathway, perhaps serving as membrane organizers.


1986 ◽  
Vol 6 (11) ◽  
pp. 3734-3745
Author(s):  
H Shida

Two classes of revertants were isolated from a vaccinia virus mutant whose hemagglutinins (HAs) accumulate on nuclear envelopes and rough endoplasmic reticulums. The HAs of one of the revertants had the same phenotype as the wild type, i.e., rapid and efficient movement to the cell surface. The HAs of the second class had biphasic transport: rapid export to the cell surface as in the wild type and slow movement to the medial cisternae of the Golgi apparatus. Biochemical and nucleotide sequence analyses showed that the HAs of all the mutants examined that have defects in transport from the rough endoplasmic reticulum to the Golgi apparatus have altered cytoplasmic domains and that the HAs of the second class of revertants lack the whole cytoplasmic domain, while the HAs of the first class of revertants have a wild-type cytoplasmic domain.


1992 ◽  
Vol 119 (6) ◽  
pp. 1459-1468 ◽  
Author(s):  
A Cooper ◽  
H Bussey

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.


Genetics ◽  
1996 ◽  
Vol 144 (2) ◽  
pp. 495-510 ◽  
Author(s):  
B K Haarer ◽  
A Corbett ◽  
Y Kweon ◽  
A S Petzold ◽  
P Silver ◽  
...  

Abstract Replacement of the wild-type yeast profilin gene (PFY1) with a mutated form (pfy1-111) that has codon 72 changed to encode glutamate rather than arginine results in defects similar to, but less severe than, those that result from complete deletion of the profilin gene. We have used a colony color-sectoring assay to identify mutations that cause pfy1-111, but not wild-type, cells to be inviable. These profilin synthetic lethal (psl) mutations result in various degrees of abnormal growth, morphology, and temperature sensitivity in PFY1 cells. We have examined psl1 strains in the most detail. Interestingly, these strains display a diploid-specific defect in bud-site selection; haploid strains bud normally, while homozygous diploid strains show a dramatic increase in random budding. We discovered that PSL1 is the late secretory gene, SEC3, and have found that mutations in several other late secretory genes are also synthetically lethal with pfy1-111. Our results are likely to reflect an interdependence between the actin cytoskeleton and secretory processes in directing cell polarity and growth. Moreover, they indicate that the secretory pathway is especially crucial for maintaining budding polarity in diploids.


Blood ◽  
2004 ◽  
Vol 103 (3) ◽  
pp. 1105-1113 ◽  
Author(s):  
Patricia Hixson ◽  
C. Wayne Smith ◽  
Susan B. Shurin ◽  
Michael F. Tosi

AbstractTwo novel CD18 mutations were identified in a patient who was a compound heterozygote with type 1 leukocyte adhesion deficiency and whose phenotype was typical except that he exhibited hypertrophic scarring. A deletion of 36 nucleotides in exon 12 (1622del36) predicted the net loss of 12 amino acid (aa) residues in the third cysteine-rich repeat of the extracellular stalk region (mut-1). A nonsense mutation in exon 15 (2200G>T), predicted a 36-aa truncation of the cytoplasmic domain (mut-2). Lymphocyte function-associated antigen 1 (LFA-1) and macrophage antigen-1 (Mac-1) containing the mut-1 β2 subunit were expressed at very low levels compared with wild-type (wt) β2. Mac-1 and LFA-1 expression with the mut-2 β2 subunit were equivalent to results with wt β2. Binding function of Mac-1 with mut-2 β2 was equivalent to that with wt β2. However, binding function of LFA-1 with the mut-2 β2 subunit was reduced by 50% versus wt β2. It was concluded that (1) the portion of the CD18 stalk region deleted in mut-1 is critical for β2 integrin heterodimer expression but the portion of the cytoplasmic domain truncated in mut-2 is not; and (2) the mut-2 cytoplasmic domain truncation impairs binding function of LFA-1 but not of Mac-1. Studies with the patient's neutrophils (PMNs) were consistent with functional impairment of LFA-1 but not of Mac-1. (Blood. 2004;103:1105-1113)


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