Structure of the yeast endoplasmic reticulum: Localization of ER proteins using immunofluorescence and immunoelectron microscopy

Preuss, Daphne; Mulholland, Jon; Kaiser, Chris A.; Orlean, Peter; Albright, Charles F.; Rose, Mark D.; Robbins, Phillips W.; Botstein, David

doi:10.1002/yea.320070902

Cited by 171 publications

(171 citation statements)

References 62 publications

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“…As expected, intact Hmg1p was present predominantly in the nuclear envelope, with increased fluorescence found in cells generating kar- Figure 5, A and B). The localization of the lumenal ER protein Kar2p demonstrates the pattern for proteins found throughout the ER (Rose et al, 1989;Preuss et al, 1991). In Figure 5, C and D, Kar2p staining appeared in the nuclear envelope and in a network stretching to the cell periphery.…”

Section: Alteration Of the Cytosolic Domain Of Hmg1p Did Not Cause MImentioning

confidence: 82%

The Role of the 3-Hydroxy 3-Methylglutaryl Coenzyme A Reductase Cytosolic Domain in Karmellae Biogenesis

Profant¹,

Roberts

Koning

et al. 1999

MBoC

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In all cells examined, specific endoplasmic reticulum (ER) membrane arrays are induced in response to increased levels of the ER membrane protein 3-hydroxy 3-methylglutaryl coenzyme A (HMG-CoA) reductase. In yeast, expression of Hmg1p, one of two yeast HMG-CoA reductase isozymes, induces assembly of nuclear-associated ER stacks called karmellae. Understanding the features of HMG-CoA reductase that signal karmellae biogenesis would provide useful insights into the regulation of membrane biogenesis. The HMG-CoA reductase protein consists of two domains, a multitopic membrane domain and a cytosolic catalytic domain. Previous studies had indicated that the HMG-CoA reductase membrane domain was exclusively responsible for generation of ER membrane proliferations. Surprisingly, we discovered that this conclusion was incorrect: sequences at the carboxyl terminus of HMG-CoA reductase can profoundly affect karmellae biogenesis. Specifically, truncations of Hmg1p that removed or shortened the carboxyl terminus were unable to induce karmellae assembly. This result indicated that the membrane domain of Hmg1p was not sufficient to signal for karmellae assembly. Using β-galactosidase fusions, we demonstrated that the carboxyl terminus was unlikely to simply serve as an oligomerization domain. Our working hypothesis is that a truncated or misfolded cytosolic domain prevents proper signaling for karmellae by interfering with the required tertiary structure of the membrane domain.

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Section: Alteration Of the Cytosolic Domain Of Hmg1p Did Not Cause MImentioning

confidence: 82%

The Role of the 3-Hydroxy 3-Methylglutaryl Coenzyme A Reductase Cytosolic Domain in Karmellae Biogenesis

Profant¹,

Roberts

Koning

et al. 1999

MBoC

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“…Bars, 0.25/~m. diminishes, and Golgi structures, followed by the endoplasmic reticulum, enter the neck and body of the bud (Preuss et al, 1991(Preuss et al, , 1992. Within such medium-sized buds (Fig.…”

Section: S Cerevisiae Cells Grow Via Localized Fusion Of Secretorymentioning

confidence: 97%

“…For these reasons, we have undertaken to study the morphology and molecular organization of the actin cables and cortical patches in the electron microscope. Previously, we developed and reported an improved immunoelectron microscopy technique that we used to identify and characterize the yeast endoplasmic reticulum and Golgi apparatus (Preuss et al, 1991 and. Further development of this method has now allowed us to visualize and identify morphological elements of the yeast actin cytoskeleton in the electron microscope.…”

mentioning

confidence: 99%

Ultrastructure of the yeast actin cytoskeleton and its association with the plasma membrane.

Mulholland

Preuss

Moon

et al. 1994

The Journal of Cell Biology

347

273

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Abstract. We characterized the yeast actin cytoskeleton at the ultrastructural level using immunoelectron microscopy. Anti-actin antibodies primarily labeled dense, patchiike cortical structures and cytoplasmic cables. This localization recapitulates results obtained with immunofluorescence light microscopy, but at much higher resolution. Immuno-EM double-labeling experiments were conducted with antibodies to actin together with antibodies to the actin binding proteins Abplp and cofilin. As expected from immunofluorescence experiments, Abplp, cofilin, and actin colocalized in immuno-EM to the dense patchlike structures but not to the cables. In this way, we can unambiguously identify the patches as the cortical actin cytoskeleton. The cortical actin patches were observed to be associated with the cell surface via an invagination of plasma membrane. This novel cortical cytoskeleton-plasma membrane interface appears to consist of a fingerlike invagination of plasma membrane around which actin filaments and actin binding proteins are organized. We propose a possible role for this unique cortical structure in wall growth and osmotic regulation.

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“…These tagged versions of wild-type and mutant desaturase alleles were fully functional as judged by the robust growth of the respective strains on media without UFAs. In vivo localization of the GFP-tagged desaturases by fluorescence microscopy revealed a staining pattern characteristic of the yeast ER, with intense labeling of both perinuclear and cortical ER domains (Preuss et al, 1991;Prinz et al, 2000; Figure 1A). Immunofluorescence analysis of the myc-tagged alleles revealed a localization similar to that observed for the GFPtagged versions (unpublished data).…”

Section: Molecular Characterization Of Ole1 Ts /Mdm2 Epitopetaggingmentioning

confidence: 99%

Lipid-dependent Subcellular Relocalization of the Acyl Chain Desaturase in Yeast

Tatzer¹,

Zellnig

Kohlwein

et al. 2002

MBoC

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The degree of acyl chain desaturation of membrane lipids is a critical determinant of membrane fluidity. Temperature-sensitive mutants of the single essential acyl chain desaturase, Ole1p, of yeast have previously been isolated in screens for mitochondrial inheritance mutants (Stewart, L.C., and Yaffe, M.P. ). J. Cell Biol. 115, 1249 -1257. We now report that the mutant desaturase relocalizes from its uniform ER distribution to a more punctuate localization at the cell periphery upon inactivation of the enzyme. This relocalization takes place within minutes at nonpermissive conditions, a time scale at which mitochondrial morphology and inheritance is not yet affected. Relocalization of the desaturase is fully reversible and does not affect the steady state localization of other ER resident proteins or the kinetic and fidelity of the secretory pathway, indicating a high degree of selectivity for the desaturase. Relocalization of the desaturase is energy independent but is lipid dependent because it is rescued by supplementation with unsaturated fatty acids. Relocalization of the desaturase is also observed in cells treated with inhibitors of the enzyme, indicating that it is independent of temperature-induced alterations of the enzyme. In the absence of desaturase function, lipid synthesis continues, resulting in the generation of lipids with saturated acyl chains. A model is discussed in which the accumulation of saturated lipids in a microdomain around the desaturase could induce the observed segregation and relocalization of the enzyme.

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Structure of the yeast endoplasmic reticulum: Localization of ER proteins using immunofluorescence and immunoelectron microscopy

Cited by 171 publications

References 62 publications

The Role of the 3-Hydroxy 3-Methylglutaryl Coenzyme A Reductase Cytosolic Domain in Karmellae Biogenesis

The Role of the 3-Hydroxy 3-Methylglutaryl Coenzyme A Reductase Cytosolic Domain in Karmellae Biogenesis

Ultrastructure of the yeast actin cytoskeleton and its association with the plasma membrane.

Lipid-dependent Subcellular Relocalization of the Acyl Chain Desaturase in Yeast

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